Neuronal growth factor galectin-1

ABSTRACT

This invention relates to a remedy for neuropathy, such as nerve injury, nerve degeneration, and hypofunction upon nerve grafting, which contains as the active ingredient galectin-1 having an amino acid sequence represented by SEQ ID NO:1 or its derivative; a protein having the amino acid sequence represented by SEQ ID NO:1 or one having a homology of 90% or more at the amino acid level with the sequence of SEQ ID NO:1 and carrying a disulfide bond(s) at least between Cys at the 16-position (Cys 16) and Cys at the 88-position (Cys 88) among cystein residues at the 2-position (Cys 2), 16-position (Cys 16), 42-position (Cys 42), 60-position (Cys 60), 88-position (Cys 88) and 130-position (Cys 130); and a process for producing the galectin-1 or its derivative protein by using an affinity column having an antibody to the above protein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to Galectin-1, or derivatives thereof,having nerve regeneration promoting activity such as the regeneration ofaxons, the repair of nerve tissues and the like, and remedies forneuropathy involving nerve injury, nerve degeneration and hypofunctionat nerve grafting which contain such proteins as the active ingredients.

BACKGROUND OF THE INVENTION

Most neuropathies, e.g. nerve injury caused by a traffic accident, nerveinjury or nerve degeneration caused by remedies for cancers or AIDS, andthe injury or hypofunction of peripheral nerves or central nerves causedby amyotrophic lateral sclerosis, diabetic neuropathy, dementia senilis,Alzheimer's disease, Parkinson's disease and the like are intractable,they present a serious condition, and often lead to death of a patient.However, at present, there is no effective remedy. Since, degenerationand deciduation of nerve tissues, transaction and regression of axons,and so on occur in these neuropathies, in order to prevent and treat theneuropathy, a factor which acts to inhibit nerve tissue degeneration orapoptosis and promoting axons regeneration, is required as an effectiveremedy.

Since Levi-Montalcini et al. found nerve growth factor (NGF) about 40years ago, it has been revealed that humoral factors acting on nervecells, i.e., a neurotrophic factor group including ciliary neurotrophicfactor (CNTF), brain-derived neurotrophic factor (BDNF), neurotrophin-3(NT-3), neurotrophin-4/5 (NT-4/5) and glia-derived neurotrophic factor(GDNF), and cytokines or the like contain a factor acting to support thesurvival of various neurons and to regenerate axons. And theapplicability of some kinds of these factors to pharmaceuticalformulations has also been studied.

Galectin is a generic name for an animal lectin specific for alactosamine sugar chain, and is also called β galactoside-binding animallectin or S-type lectin. Galectin is confirmed to exist in the cells ofanimal tissues ranging from lower Invertebrata such as nematode andsponge to bird and human. Some species of this type of lectin had beenfound, and in 1994, these were proposed to be generically called“galectin” (S. H. Barondes et al, Cell, 76, 597-598, 1994). Up untilnow, galectin-1 to galectin-11 have been reported as members of thegalectin family. It has been reported that the actions of thesegalectins are associated with cell growth and cell adhesion, but thesephysiological functions are still unknown (J. Hirabayashi et al., J.Biochem., 119, 1-8, 1996; N. L. Perillo et al., J Mol Med., 76, 402-412,1998). With regard to galectin-1, the structures of a large number ofanimal-derived forms have been determined (human-galectin-1: J.Hirabayashi et al., J. Biochem., 104, 1-4, 1988; J. Hirabayashi et al.,Biochim. Biophys, Acta., 1008, 85-91, 1989, rat galectin-1: L. B. Clerchet al., Biochemistry, 27, 692-699, 1988, mouse galectin-1: T. J. G.Wilson et al., Biochem. J., 261, 847-852, 1989, bovine galectin-1: W. M.Abbot et al., Biochem. J., 259, 283-290, 1989). Information regardingthe galectin-1 gene and protein, and a remedy for autoimmune diseasesare disclosed in Japanese Patent Application Laying-Open (kokai) No.2-84192 (Title: Mammalian 14-β-gal Lectins, Applicant: IdeonCorporation) and International Publication WO94/11497 (Title: Method ofcausing selective immunosuppression using HL-60-related lectins,Applicant: Incyte Pharmaceuticals Inc.) However, there is no descriptionregarding galectin-1 as a remedy for neurodegenerative diseases such asnerve injury and neuropathy in the above documents.

On the other hand, as stated below, there are some reports on therelation between galectin-1 and the nervous system: galectin-1 expressesin a dorsal spinal nerve root ganglion cell at the development stage ofa sensory nerve (J. Dodd, et al. J Exp Biol. 124, 225-238, 1986; M. A.Hynes, et al. J. Neurosci., 10, 1004-1013, 1990); galectin-1 isassociated with the aggregation of nerve cells or the development ofneurites, as a cell adhesive substrate, in a dorsal spinal root ganglioncell (R. L. Outenreath et al., J. Neurocytol., 21, 788-795, 1992);galectin-1 expresses in a rat olfactory nerve cell at the developmentstage, and is associated with the development of axons as a celladhesive substrate (N. K. Mahanthappa, et al. Development. 120,1373-1384, 1994; E. H. Raabe, et al. Brain Res Dev Brain Res. 101,187-196, 1997); galectin-1 is associated with the development of axonsin a culture system comprising mouse olfactory nerve cells, as a celladhesive substrate (A. C. Puch, et al. Dev. Biol. 179, 274-287, 1996);there are reports on the distribution of galectin-1 in some kinds ofnerve tissues (R. Joubert et al, Dev. Neurosci. II, 397413, 1989; S.Kuchler et al, Dev. Neurosci., II, 414-427, 1989), etc. These reports ongalectin-1 are limited to discussion of its distribution in nervetissues or its function as an adhesive substrate of nerve cells, andcontain no description of galectin-1 as a factor that promotes nerveregeneration by acting on a nerve cell or a paraneural system, such asdo neurotrophic factors or cytokines which act on the nervous system.Furthermore, it has been found that human galectin-1 has 6 cysteineresidues in a molecule thereof, and that this protein has β-galactosidebinding activity in a state where this protein is reduced, i.e., thecysteine is free, whereas this protein does not have that activity in astate where this protein is oxidized, i.e., a SS bond is formed. All ofthe above-mentioned laying-open applications or reports regarding thenervous system describe regarding the existence and function ofgalectin-1 as a lectin. However, there is no report at all in respect ofthe physiological action of galectin-1 on nervous systems as a nerveregeneration promoting factor or a nerve survival-support factor in astate where this protein is oxidized, i.e., SS bonds are formed andthere is no lectin activity.

Not only the above stated biological properties, but alsophysicochemical properties are also greatly different between oxidizedgalectin-1 and reduced galectin-1. Since the formation of a SS bond byoxidation means that one cystine residue is formed from two cysteineresidues, accordingly the oxidized protein loses two hydrogen atoms perSS bond, i.e., 2 Dalton (Da) of molecular weight are decreased. Humangalectin-1 may form 3 couples of SS bond, and in such a case, theprotein loses 6 Da of molecular weight. The difference of molecularweight can be determined by measuring the molecular weight with a highprecision mass spectrometer. Furthermore, since the higher orderstructure of protein is greatly changed by the formation of SS bond, andboth the steric dimensions of molecule and amino acid residues existingon a molecular surface are changed, the mobility on SDS electrophoresisand the elution time in reversed phase chromatography, ion exchangechromatography and the like are also changed. By examiningphysicochemical properties, it is possible to distinguish whethergalectin-1 is in an oxidized state or in a reduced state.

In neuropathy, the degeneration and deciduation of nerve tissues, thetransection and regression of axons, and the like occur. So, as aneffective remedy for preventing or treating this disease, a neurotrophicfactor or the like which acts to control the degeneration of nervetissues or apoptosis and promote the regeneration of neurites isdesired. The study on the applicability of a neurotrophic factor groupincluding NGF, CNTF, BDNF etc. to remedies has been proceeding. Theseneurotrophic factor groups are ones having a function of promotingneurite regeneration and supporting the survival, and these were mainlyfound in nerve cells isolated from fetal or young animals at thedevelopment stage. So, to allow expression of the actions ofneurotrophic factors, it is necessary to allow the factors to directlyact on nerve cells. However, the reactivity to factors may be differentbetween nerve cells of adult animals and those of fetal or younganimals. Furthermore, as distinct from its state in a culture plate,each nerve cell does not exist separately in vivo, but generally nervecells attach with each other or are enclosed by paraneural system cellssuch as Schwann cell, so that they exchange information interactively tomaintain their functions. Where nerves are regenerated after nerveinjury occurred, nerve cells perform cross talk with other cellssurrounding them to repair their functions. As is clear from the abovedescriptions, there is the problem of how the factor groups can be madeto act on nerve cells on which they directly act, in other words, thereis the problem of administration method, therefore the development ofremedies for neuropathy faces difficulty.

Under these circumstances, using the organ culture system of nervetissues which maintains its functional structure in vivo, the presentinventors have continued a thorough study to find out a novel proteinfactor indicating activity which promotes neurite regeneration from atransected nerve end of nerve tissues and supports its survival, or anovel use of the known factors as remedies for treating neuropathy ornerve injury.

To sum up, the main purpose of the present invention is to providegalectin-1 which is effective for treating neuropathy involving nerveinjury, nerve degeneration and hypofunction at nerve grafting andderivatives thereof, and remedies for neuropathy containing them asactive ingredients.

SUMMARY OF THE INVENTION

The present inventors have carried out various studies to obtain afactor promoting neurite regeneration from a transected nerve end ofnerve tissues and supporting (or maintaining) the survival thereof. Asan evaluation method which is much similar to in vivo conditions, anorgan culture assay system was applied, wherein the dorsal root gangliontissues of an adult or aged rat were embedded into a collagen gel, andthe axonal regeneration from the transected nerve end was observed,therewith the factor was screened. As a result, from the COS1 cellculture supernatant into which cDNA derived from rat liver wastransfected with an animal expression vector, an active factor promotingthe axonal regeneration from a transected nerve end of nerve tissues andsupporting the survival thereof, was purified. Then, a part of thesequence was determined, and this factor was identified as a proteinhaving galectin-1 sequence. Furthermore, it was also found that aprotein expressed from DNA encoding the galectin-1 promotes the axonalregeneration both in vitro and in vivo and has survival supportingactivity.

The inventors have conducted various studies to obtain a factor, whichpromotes regeneration of axons from the transected nerve fiber terminalof the nerve tissue and maintains its survival. The inventors havescreened for such a factor by an organ culture assay system comparableto in vivo conditions. In this assay system, DRG (dorsal root ganglion)derived from a mature or aged rat was embedded in collagen gel, andregeneration of axons from the transected nerve end was observed. Thus,the inventors have purified an active factor, which promotesregeneration of axons from the transected nerve end of DRG and maintainssurvival of the nerve fiber, from the culture supernatant of COS 1cells; determined its partial sequence; and identified it as a proteinhaving galectin-1 sequence. In addition, the inventors have found that aprotein expressed by DNA encoding this galectin-1 can promoteregeneration of axons both in vitro and in vivo, that is, it hassurvival-maintaining activity of the nerve fiber.

The present invention provides remedies for neuropathy involving nerveinjury, nerve degeneration, and hypofunction (or dysfunction) at nervegrafting, which contain as an active ingredient galectin-1 or itsderivatives having an amino acid sequence shown in SEQ ID No:1.

Galectin-1 or its derivatives used in this invention may have lectinactivity, or may have almost no lectin activity or no lectin activity.As used herein, the term “lectin activity” means β-galactoside bindingactivity. Lectin having such activity normally possesses an ability tobind to a lactose column or an ability to allow hemagglutination.

In an embodiment of this invention, galectin-1 or its derivatives carrya disulfide bond(s) at least between Cys at the 16-position (Cys 16) andCys at the 88-position (Cys 88) among cysteine residues at the2-position (Cys 2), 16-position (Cys 16), 42-position (Cys 42),60-position (Cys-60), 88-position (Cys 88) and 130-position (Cys 130) inthe amino acid sequence shown in SEQ ID NO:1.

As used herein, the term “oxidized” means that two or more cysteineresidues of the protein are in an oxidized state, that is, the residuesform a disulfide bond(s).

The protein of this invention possesses a nerve regeneration-promotingeffect, including regeneration of axons and repair of nerve tissues. Inthis respect, the protein of this invention has a function similar tothat of a neurotrophic factor. A known type of galectin-1 is a reducedtype, has lectin activity, and has an effect as an adhesive substrate ofnerve cells (N. K. Mahanthappa et al., supra; A. C. Puch et al., supra).However, it was not known that galectin-1 functions as a nerveregeneration-promoting factor.

Galectin-1 or its derivatives illustrated according to an embodiment ofthis invention, carry disulfide bonds between the following Cys residuesin the amino acid sequence shown in SEQ ID NO:1:

-   (1) Cys 16-Cys 88, Cys 2-Cys 130 and Cys 42-Cys 60; or-   (2) Cys 16-Cys 88, Cys 2-Cys 60 and Cys 42-Cys 130; or-   (3) Cys 16-Cys 88, Cys 2-Cys 42 and Cys 60-Cys 130, or    they may comprise a mixture of at least two groups out of (1), (2)    and (3), especially the one contains 50% or more said (1).

Examples of the derivatives are as follows:

-   (a) Derivatives, which have an amino acid sequence comprising at    least one amino acid substitution, deletion, insertion and/or    addition relative to the amino acid sequence shown in SEQ ID NO:1,    and have a nerve regeneration-promoting activity; or have an amino    acid sequence substantially shown in SEQ ID NO:1. The term    “substantially” means that such an amino acid sequence can contain a    change (i.e., substitution, deletion, insertion and/or addition) in    at least one amino acid residue that has no effect on nerve    regeneration-promoting activity.-   (b) Derivatives, which have a homology of 80% or more, preferably    90% or more, more preferably 95% or more at amino acid level with    the amino acid sequence shown in SEQ ID NO:1.-   (c) Derivatives, which have the acylated (for example, formylated    and acetylated) N-terminal end.-   (d) Derivatives, in which Met⁻² Lys⁻¹ or Met⁻¹ is added to the    N-terminal end.-   (e) Derivatives, which are covalently bound with a water-soluble    polymer (for example, polyethylene glycol) or a carbohydrate    chain(s).

Remedies of this invention are useful in treatment for neuropathy, forexample, promotion of nerve regeneration and functional recovery fromcentral and peripheral nerve damage due to external injuries resultedfrom an accident and surgery; disorders resulted from nerve damages dueto curative treatment, such as chemotherapy or radiation therapy againstdiseases including cancer, AIDS, and the like; nerve damages resultedfrom central and peripheral nerve injuries due to drugs, heavy metals,chemicals such as alcohols; nerve injuries resulted from ischaemia orinfection, malignancy or metabolic disorders, for example, diabeticneuropathy, or dysfunction in kidney or liver; degeneration of specialnerve cells including amyotrophic lateral sclerosis, which is a motornerve degenerative disorder, and nerve degenerative disorders, such asAlzheimer's disease. Moreover, the remedies of this invention can beused as a nerve regeneration-promoting agent for recovery of neuropathysuch as hypofunction upon nerve grafting.

Remedies of this invention can be made into a pharmaceutical form, suchas for oral and parenteral administration by combining with apharmaceutically acceptable liquid or solid carrier. In addition, theremedy may contain one or more factors having neurotrophic activity,including NGF (nerve growth factor), and BDNF (brain-derived nervegrowth factor); or extracellular matrix having such factors orparaneural cells.

In an embodiment of this invention, the remedies of this invention maybe in the form that is prepared by allowing the protein of thisinvention to be contained into collagen gel, adding (an)otherneurotrophic factor(s) if necessary, and directly embedding at a site ofnerve injury. In this case, essential ingredients, such aspharmaceutical agent and carrier, are placed or packed within a tubemade of a biocompatible material (for example, silicon rubber, collagen,polypropylene, polyester, or polyamide).

Moreover, the present invention relates to a method for the treatment ofneuropathy, comprising administering the above remedy of this inventionto a patient (including human) who needs the treatment of neuropathysuch as nerve injury, nerve degeneration, or hypofunction upon nervegrafting.

The present invention also provides the above-defined galectin-1 or itsderivatives.

Furthermore, the present invention provides a process of producing theabove protein comprising the steps of loading a substance containing theabove-defined galectin-1 or its derivative (for example, a naturalsubstance or the one prepared by recombination or a chemical method) toan affinity column to which an antibody or antibodies to the aboveprotein are bound, allowing the protein to be adsorbed, subsequentlyeluting the protein, and if necessary oxidizing the protein.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 is a photograph of an electrophoresis showing the results ofNorthern Blot assay regarding RNA obtained from each tissue of a rat.

FIG. 2 is a photograph of an electrophoresis image of the nerveregeneration promoting factor purified from the culture supernatant ofpRLF transfected COS1 cells.

FIG. 3 is a peptide map determined by a reversed phase chromatography,after digesting the nerve regeneration promoting factor purified fromthe culture supernatant of pRLF transfected COS1 cells with lysylendopeptidase.

FIG. 4 shows the results regarding the nerve regeneration promotingactivity of an Escherichia coli expression product Gal1 (1-134). Herein,“Transected central end” and “Transected peripheral end” refer to “atransected central nerve end” and “a peripheral nerve end”,respectively.

FIG. 5 is a peptide map separated by a reversed phase chromatography,after digesting an Escherichia coli expression product Gal1 (1-134) withtrypsin.

FIG. 6 is a peptide map separated by a reversed phase chromatography,after the secondary digesting TP9, the tryptic digestion fragment of anEscherichia coli expression product Gal1(−134), with lysylendopeptidase.

FIG. 7 shows the results of examining hemagglutination activity of anEscherichia coli expression product Gal1 (1-134).

FIG. 8 is two electron micrographs showing a peripheral site positionedat 6 mm from the crush lesion site into which Gal1 (1-134) (A) and acontrol (B) were respectively administered for 14 consecutive days.

FIG. 9 is two photographs showing HE-stained images of longitudinalfrozen nerve sections from rat, which were subjected to aperfusion-fixation 10 days after operation of administering Gal1 (1-134)(A) and a control (B) respectively.

FIG. 10 is two photographs showing immuno-stained images with an anti-NFantibody of longitudinal frozen sections from rat, which were subjectedto a perfusion-fixation 10 days after operation of administering Gal1(1-134) (A) and a control (B) respectively.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention is further described with regard tothe method of producing the protein of the present invention which has anerve regeneration promoting activity (hereinafter “the protein of thepresent invention”) and pharmaceutical compositions containing theprotein.

Gene Construction

The protein of the present invention can be obtained by a process of:constructing a recombinant vector comprising DNA encoding a complete orpartial amino acid sequence shown in SEQ ID NO:1, or DNA encoding thederivatives of the above amino acid sequence (e.g. an amino acidsequence wherein one or more amino acids are substituted, deleted,inserted and/or added); transforming a host cell with the above vector,culturing the obtained host cell; and finally separating and purifyingthe protein of interest.

The DNA encoding the protein of the present invention can be obtained bya process of: obtaining DNA by the restriction enzyme cleavage ofgenomic DNA, cloning from cDNA library, or DNA synthesis, and thenconverting and amplifying the obtained DNA by site-directed mutagenesistechniques such as oligonucleotide site-directed mutagenesis or cassettemutagenesis, or PCR method. In this case, for example, a techniquedescribed in Molecular Cloning (Sambrook et al., Cold Spring HarborLaboratory Press (1989)) can be applied.

The gene of the protein of the present invention and its structure arealready known in respect of various living things including human andmouse (e.g. Abbott et al., Biochem. J., 259, 291-294, 1989; Chiariottiet al., Biochim. Biophys. Acta, 1089, 54-60, 1991). So, on the basis ofthe information regarding these known nucleotide sequences and aminoacid sequences, DNA encoding the protein of the present invention can beobtained/produced from cDNA library, as appropriate, using PCR method,DNA synthesis technique and so on.

As shown in Examples described later, the cDNA encoding the protein ofthe present invention can be obtained by producing a cDNA library fromhuman liver tissue in accordance with standard techniques and thenapplying PCR method, using primers produced on the basis of the knownnucleotide sequence of human galectin-1.

In the case of applying DNA chemical synthesis, according to e.g. themethod of Alton et al. (Japanese Patent Application Laying-Open (kohyo)No. 59-501097), a DNA fragment encoding the protein of the presentinvention can be obtained by designing the nucleotide sequence on thebasis of the amino acid sequence of the protein of the presentinvention, and using preferential codons, if necessary.

Furthermore, regarding a polypeptide derived by deletion, addition,insertion and/or substitution of one or more amino acid residues in theamino acid sequence shown in SEQ ID NO: 1, on the basis of theabove-stated DNA encoding galectin-1, DNA encoding the mutantpolypeptide can be produced by site-directed mutagenesis techniques suchas oligonucleotide site-directed mutagenesis or cassette mutagenesis(e.g. Mark et al., Proc. Natl. Acad. Sci. USA, 81, 5662-5666, 1984;Inouye et al., Proc. Natl. Acad. Sci. USA, 79, 3438-3441, 1982; PCTWO85/00817 laid-open on Feb. 28, 1985; and Wharton et al., Nature, 316,601-605, 1985) or PCR method, or by DNA chemical synthesis.

Applicable host cells include prokaryotic cells (e.g. those of bacteriaand preferably Escherichia coli) and eukaryotic cells (e.g. those ofyeast, insect or mammal). Examples of mammalian cells include a COScell, Chinese Hamster Ovary cell, X63.6.5.3. cell, C-127 cell, BHK (BabyHamster Kidney) cell, human-derived cell (e.g. HeLa cell), etc. Examplesof yeast include a baker's yeast (Saccharomyces cerevisiae),methanol-assimilated yeast (Pichia pastoris), etc. Examples of insectcells include a silk worm culture cell (e.g. Sf21 cell) and the like.

When the protein of the present invention is produced using aprokaryotic or eukaryotic cell, the protein can be obtained by a processof: adding cleavage sites by restriction enzymes and/or promoter DNAfacilitating expression, to DNA encoding the protein; integrating theabove DNA into an appropriate expression vector, culturing the cellstransformed or transfected by the vector, and separating and purifyingthe generated protein of the present invention. In the case whereEscherichia coli is selected as a host, a codon (priority codon) whichis preferable to the expression in Escherichia coli may be integrated.

Vectors used for transforming Escherichia coli include pKC30 (ShimatakeH. and M. Rosenberg, Nature, 292, p 128-132, 1981), pTrc99A (Amann E. etal, Gene, 108, 193-200, 1991), pCFMS36 (ATCC No. 39934; Japanese PatentApplication Laying-Open (kohyo) No. 60-501988), etc.

Vectors for mammalian cells include pSV2-neo (Southern and Berg, J. Mol.Appl. Genet., 1, 327-341, 1982), pCAGGS (Niwa et al., Gene, 108,193-200, 1991) or pcDL-SR α 296 (Takebe et al., Mol. Cell. Biol., 8,466-472, 1988), etc. Those for yeast include pG-1 (Schena M. andYamamoto K. R., Science, 241, 965-967, 1988), etc. Those for silk wormcells include a transfer vector pAc373 used for preparing recombinantviruses (Luckow et al., Bio/Technology, 6, 47-55, 1988), etc.

These vectors may comprise a replication origin, selective marker,promoter and ribosome binding-site, as needed, and these for eukaryoticcells may further comprise an RNA splice site, polyadenylation signaland so on, as needed.

Examples of replication origins comprised in vectors for mammalian cellsinclude those derived from SV40, adenovirus, bovine papilloma virus,etc. Examples of replication origins comprised in vectors forEscherichia coli include those derived from ColE1, R factor, F factor,etc. Replication origins comprised in vectors for yeast include thosederived from 2 μm DNA, ARS1, etc.

Promoters for gene expression comprised in vectors for mammalian cellsinclude those derived from viruses, e.g. retrovirus, polyoma virus,adenovirus, SV40, etc., or those derived from chromosomes, e.g. EF1-α,etc. The promoters comprised in vectors for Escherichia coli include apromoter derived from bacteriophage λ or a trp, lpp, lac or tacpromoter, etc. The promoters comprised in vectors for baker's yeastinclude an ADH, PH05, GPD, PGK or MAF α promoter, and those formethanol-assimilated yeast include AOX1 promoter and the like. Thepromoters for silk worm cell vectors include that derived from nuclearpolyhedrosis virus and the like.

Selective markers for vectors for mammalian cells include a neomycin(neo) resistance gene, thymidine kinase (TK) gene, dihydrofolatereductase (DHFR) gene, Escherichia coli xanthine guaninephosphoribosyltransferase (Ecogpt) gene, etc. Those for vectors forEscherichia coli include a kanamycin resistance gene, ampicillinresistance gene, tetracycline resistance gene, etc. Those for vectorsfor yeast include a Leu2, Trp1 or Ura3 gene, etc.

For example, when the protein of the present invention consisting ofamino acids 1 to 134 of the amino acid sequence shown in SEQ ID NO:1 isproduced, first, DNA encoding amino acids 1 to 134 is synthesized, andthen NcoI site (which comprises ATG of initiation codon) is added toN-terminal of the DNA, whereas stop codon is added to the C-terminal andBamHI site is added downstream of the C-terminal. This DNA fragment istreated with NcoI and BamHI, and linked to pET-3d (Stratagene) which wasdigested with NcoI and BamHI. Then, using Epicurian Coli BL21 (DE3)Competent Cells (Stratagene), a transformant used for expressing theprotein of the present invention is obtained from Escherichia colihaving an expression vector. In an expression plasmid pET-3d, the geneof interest is inserted downstream of T7 phage promoter and transcribedby T7 RNA polymerase provided from a host Escherichia coli. Since thisT7 RNA polymerase gene is integrated into a host Escherichia colichromosome which is downstream of lac UV5 promoter, its expression canbe controlled by inducing with the addition of IPTG.

Protein Expression, Refolding and Purification

In order to obtain the protein of the present invention using theabove-described host-vector systems, the following process may beapplied: a host cell is transformed by a recombinant DNA wherein thegene is integrated into the proper site of the above vectors; theobtained transformant is cultured; and the polypeptide is separated andpurified from cells or culture medium. This process can be carried outby the combined use of known means and methods.

In the case of expressing the protein using a host, the original signalsequence may be converted, or the signal sequences of other proteins maybe used, in order to more reliably uniformizing the N-terminus of theexpression product. Or, N-terminus can be uniformized even by converting(substituting or adding) amino acid residues positioned at, or in thevicinity of the N-terminus (e.g. adding arginine or lysine residuesother than methionine residues, in the case of expressing the proteinusing Escherichia coli).

Furthermore, the protein of the present invention can also be obtainedby a process of: adding Glutathione-S-transferase (GST), histidine tag,FLAG peptide or the like to the N- or C-terminus of the protein of thepresent invention through the recognition peptide of a specific enzyme(e.g. thrombin, Factor Xa, enterokinase, etc.); expressing as a fusionprotein in an appropriate host and isolating it; and then treating thefusion protein with an applicable enzyme.

An example of the protein of the present invention includes a proteincomprising an amino acid sequence shown in SEQ ID NO:1. In addition, thepresent invention even comprises the derivatives of the protein of thepresent invention which comprise a conversion (i.e., a substitution,deletion, insertion and/or addition) in a portion of the amino acidsequence.

Examples of protein derivatives of the present invention can haveimproved stability or persistence in vivo, and reduced immunogenicity bye.g. a conversion (a substitution, deletion, insertion and/or addition)of amino acids, the acylation of N-terminus, and the binding of watersoluble polymers such as polyethylene glycol to α-amino group or ε-aminogroup.

As a general method of improving the thermodynamic stability of protein,it has been theoretically shown that the introduction of proline residueand the removal of glycine residue are effective (Matthews et al., Proc.Natl. Acad. Sci. U.S.A. 84, 6663-6667, 1987). Thus, to improve thestability of the protein derivatives of the present invention, designinga protein derivative into which a proline residue is introduced and aprotein derivative where glycine residue is removed, has beenconsidered.

Since the comformation of a protein is generally formed so that theprotein has hydrophobic amino acids inside and hydrophilic amino acidsoutside, the improvement of the solubility of the protein can beexpected by substituting amino acids existing on the protein surface bymore hydrophilic charged amino acids. Also the protein derivatives ofthe present invention can be designed from this point of view.

In addition, there can also be applied the amino acid sequences ofgalectin-1 from other species having homology with human galectin-1(e.g. chicken galectin: Ohyama, Y et al., Biochem. Biophys, Res. Commum.vol.134, p. 51-56 (1986); rat galectin: Clerch, L. B. et al.,Biochemistry, vol.27, p. 692-699 (1988), mouse galectin: Wilson, T. J.G. et al., Biochem. J. vol.261, p. 847-852 (1989), bovine galectin:Abbot, W. M. et al., Biochem. J. vol.259, p. 283-290 (1989), etc.) Forexample, comparing amino acid sequences between human and rat, there canbe selected either a site which has proline for rat, but an amino acidother than proline for human, or another site which has glycine forhuman, but an amino acid other than glycine for rat.

Furthermore, the proteins of the present invention include the humanprotein of the present invention having an amino acid sequence shown inSEQ ID NO:1 or derivatives thereof, wherein lysine and methionineresidues are added to the positions −1 and −2, or wherein a methionineresidue is added to the position −1. It is known that, when anexpression is carried out in a cell using bacteria (e.g. Escherichiacoli) as a host, there may be obtained a protein, wherein an initiationmethionine residue is added to the N-terminal side of protein having anerve regeneration promoting activity. In addition, depending on hosts,the generated protein with a nerve regeneration promoting activity maybe glycosylated, or the N-terminus of the generated protein may or maynot be blocked by an acetyl residue, formyl residue or the like. Theprotein of the present invention also comprises these kinds of proteins.

The protein of the present invention may be purified and isolated fromnatural sources (e.g. a conditioned medium having a nerve regenerationpromoting activity, or human lung, kidney, placenta, etc.), but it ispreferably obtained by gene recombination. With the latter method, thereis the advantage that mass production is possible.

When the protein of the present invention is purified from naturalsources or recombinant cells, one or more general protein purificationmethods set forth below can be used in combination: salting out,ammonium sulfate fractionation, solvent extraction, HPLC, affinitychromatography, ion exchange chromatography, lectin affinitychromatography, pigment adsorption chromatography, hydrophobicinteraction chromatography, gel filtration chromatography, reversedphase chromatography, heparin affinity chromatography, sulfated gelchromatography, hydroxyl apatite chromatography, metallic chelatingchromatography, isoelectric chromatography, preparative scaleelectrophoresis, isoelectric focusing method and so on. Otherwise, thephysicochemical properties of the protein of the present invention,which can be assumed from Examples described later, can also be used. Inaddition, an antibody column using an antibody capable of recognizingthe protein of the present invention can also be used.

Regarding the amino acid sequence of the protein of the presentinvention comprising 6 cysteine residues, it is desirable that it existsin a state where it is cross-bridged (oxidized) with a disulfidebond(s). The oxidation methods of converting a reduced protein to anoxidized protein include a chemical oxidation method or a disulfideexchange reaction. The chemical oxidation methods include an airoxidation method, an air oxidation method wherein heavy metallic ion(e.g. Cu²⁺) is used as a catalyst, a method wherein iodosobenzoic acid,hydrogen peroxide or the like is used, and so on. A typical example ofdisulfide exchange reaction is a method wherein a redox buffercontaining both reduced and oxidized glutathiones is used, but a methodwherein a redox buffer containing cysteine, dithiothreitol,2-mercaptoethanol and cysteamine can also be used. Thus, the protein ofthe present invention having a disulfide bond(s) at least between Cys onthe 16-position (Cys16) and Cys on the 88-position (Cys88) amongcysteine residues on the 2-position (Cys2), 16-position (Cys16),42-position (Cys42), 60-position (Cys60), 88-position (Cys88) and130-position (Cys130) can be obtained. The protein comprises 1 to 3disulfide bond(s), preferably 2 to 3 disulfide bonds, and morepreferably 3 disulfide bonds. When the refolding of protein is carriedout expressing DNA encoding an amino acid sequence of SEQ ID NO:1 inEscherichia coli, the protein is obtained as a mixture which consists ofthe following 3 types of oxidized galectin-1 having a disulfide bondbetween each cysteine: (1) Cys16-Cys88, Cys2-Cys130 and Cys42-Cys60; (2)Cys16-Cys88, Cys2-Cys60 and Cys42-Cys130; and (3) Cys16-Cys88,Cys2-Cys42 and Cys60-Cys130, and further comprises more than 50% ofgalectin-1 of the above (1). When the above DNA is expressed in a COS1cell, the oxidized galectin-1 of the above (1) is mainly obtained. Whena high-performance reversed phase chromatography, wherein acetonitrileconcentration in 0.1% TFA increases linearly 32% to 40% for 60 minutesat room temperature, is carried out using YMC Protein RP (10 mm×250 mm,YMC) as a column, the oxidized galectin-1 expressed in Escherichia coliis eluted, as approx. 36% acetonitrile concentration (in the case ofform (1)) and approx. 34% acetonitrile concentration (in the case offorms (2) and (3)). When a high-performance reversed phasechromatography, wherein acetonitrile concentration in 0.1% TFA increaseslinearly 32% to 44% for 45 minutes at room temperature, is carried outusing YMC Protein RP (4.6 mm×150 mm, YMC) as a column, in the case ofform (1), the oxidized galectin-1 expressed in a COS1 cell is eluted asapprox. 38% acetonitrile concentration.

Pharmaceutical Composition

The present invention includes remedies for neuropathy involving nerveinjury, nerve degeneration, hypofunction at nerve grafting and so onwhich contain as an active ingredient the above-defined galectin-1 orits derivatives.

The remedies may include a diluent, antiseptic, solubilizer, emulsifieror adjuvant, as well as a general appropriate liquid or solid carrier(Remington: The Science and Practice of Pharmacy, Nineteenth Edition,Mack Publishing Company, 1995). Such a pharmaceutical composition has aliquid or solid form, and is able to be mixed with a diluent selectedfrom buffers having various pH and ionic strength (e.g.Tris-hydrochloric acid, acetate, phosphate), an additive such as albuminor gelatin for preventing surface adsorption, a surfactant (e.g. Tween20, Tween 80, Pluronic F68, bile salt), a solubilizer (e.g. glycerol andpolyethylene glycol), an antioxidant (e.g. ascorbic acid and sodiummetabisulfite), an antiseptic (e.g. thimerosal, benzyl alcohol,paraben), and an excipient or an isotonizing agent (e.g. lactose andmannitol).

Furthermore, the properties of the pharmaceutical composition of thepresent invention include a covalent bond between the protein and awater soluble polymer such as polyethylene glycol; chelate complexationwith metallic ions; or the uptake of the above substance into a granularformulation of polymerized compound such as polylactate, polyglycolateor hydrogel, or onto its surface, or into a liposome, microemulsion,micelle, monolayer- or multilayer-caveola, erythrocyte ghost, orspheroplast. The composition having the above-stated characteristics hasan influence on the physical condition, solubility, stability, in vivorelease rate, and in vivo clearance of the protein of the presentinvention. So, the composition is selected depending on the physical andchemical properties of the protein having nerve regeneration promotingactivity.

The remedies of the present invention can have various administrationroutes including parenteral, transpulmonary, transnasal, peroral orlocal imbedding administration. And, depending on these administrationroutes, processes such as granulation, protective coating, mixing withprotease inhibitor, mixing with absorbefacient, enclosure in biologicalmaterials or biocompatible materials such as collagen can be applied tothe remedies. The dosage forms include solution, suspension, emulsion,tablet, pill, capsule, aerosol, enteric coated tablet, sustained releasepreparation, imbedding preparation and the like, but are not limitedthereto. In the embodiment of the present invention, the protein of thepresent invention may be contained in collagen, to directly be imbeddedinto a neurological location, but for example, necessary ingredientssuch as agent or carrier can be included into a tube made of abiocompatible material (e.g. silicone rubber, collagen, polypropylene,polyester, polyamide, etc.)

Regarding remedies comprising the protein of the present invention,generally 0.01 μg/kg body weight to 1 mg/kg body weight can beadministered as an active ingredient one to several times per day,according to age, body weight, symptom, sex, administration route and soon. However, the dosage is not limited to the above range, and it canvary on various therapeutical factors.

The combined use of the protein of the present invention with a singleor other additive neurotrophic activity factor groups is useful fortreating a large number of nervous system disorders. Other additivefactors include a member of the neurotrophin family including NGF, BDNF,NT-3, NT-4/5, NT-6 and the like; the insulin family including insulin,IGF-I, IGF-II and the like; the FGF family including aFGF, bFGF, FGF-5and the like; the interleukin group including IL-1, IL-2, IL-3, IL-6 andthe like; and LIF, GM-CSF, G-CSF, EPO, TPO, CNTF, oncostatin M, TNF α,thioredoxin, GDNF, TGF β, EGF, growth promoting activity, growthinhibitory factor, plasminogen, glia-derived nexin, α₂ macroglobulin,S100 protein, annexin V, neuron specific enolase, thrombospondin, andhepatocyte growth factor. Furthermore, neuro-peptides includingganglioside such as GM1 and GM2, adrenocorticotropic hormone (ATCH),thyrotropin-releasing hormone (TRH), hippocampal cholinergicneurotrophic peptide (HCNP), corticotropin-releasing hormone (CRF) andthe like are also included to the protein of the present invention.Among them, NGF, BDNF, NT-3, NT-4/5, NT-6, IGF-I, IGF-II, CNTF and GDNFare desirable.

Moreover, the combined use of the protein of the present invention withan extracellular matrix or paraneural system cell is also useful fortreating nervous system disorders. Extracellular matrixes includelaminin, fibronectin, thrombospondin, collagen and the like. Paraneuralsystem cells include Schwann cell, fibroblast, satellite cell,microphage, glia cell and the like. In addition, the combined use ofparaneural system cells with basal membranes is also useful for treatingnervous system disorders.

According to in vitro and in vivo experiments, the protein of thepresent invention has been proved to promote axonal regeneration andremyelinization from nerve injuries due to denervation, crush, freezingof nerves. The system wherein a dorsal root ganglion associated a nervestumps was embedded into a collagen gel and the effect of axonalregeneration from a nerve stumps was examined (Horie H. et at. NeurosciLett 121, 125-128 (1991); Horie H. et al. NeuroReport 2, 521-524 (1991))showed a clear axonal regeneration effect. Further, even in vivo, therewas shown a nerve regeneration effect from nerve injuries due to thedenervation, crush and freezing of an sciatic nerve. Referring topreviously reported methods (S. Varon, et at, pp. 101-122 in “Frontiersof clinical neuroscience, vol.6 Neural Regeneration and Transplantation”edited by F. J. Seil (Alan R Liss, Inc.) (1989); G. Lundborg, et al.,Exp. Neurol., 76, 361-375 (1982); L R Williams, et al, J. Comp. Neurol.,218, 460-470 (1983); Q. Zao, et al., Restor. Neurol, Neurosci., 5,197-204 (1993)), a silicon chamber was attached to an sciatic nerve, andaxonal regeneration in the chamber was observed. As a result of thisexperiment, it was shown that the protein of the present inventionpromotes axonal regeneration in the chamber filling collagen gels.Furthermore, as a result of another experiment which involved inflictinginjuries, including crushing and freezing, to an sciatic nerve, followedby observing the damaged part with an electron microscope (A Seto,Hasegawa M, Uchiyama N, Yamashima T, Yamashita J: J Neuropathol ExpNeurol 56:1182-1190 (1997)), it was clear that the administration of theprotein of the present invention promotes axonal regeneration andremyelinization. From the results of these experiments regarding thenerve regeneration promoting effect, the protein of the presentinvention is considered to be useful for treating neuropathy involvingvarious nerve injuries, nerve degeneration and the like, which areattended with the regression or demyelination of axons.

The main uses of the protein of the present invention are the promotionof nerve regeneration and function recovery from central and peripheralnerve injuries suffered from an accidental injury or surgical operation.Or, another use of the protein is treating for nerve injuries sufferedas a result of curative treatments such as chemotherapy and radiationtreatment for cancers, AIDS and the like. In addition, this protein canbe administered to neuropathies which are caused by central andperipheral nerve injuries suffered from chemical substances such asagent, heavy metal and alcohol. Such neuropathies may be caused by nerveinjuries which are suffered as a result of ischaemia, infection,malignant tumor or metabolic disorder, e.g. diabetic neuropathy or thedysfunction of kidney or liver. Further, such neuropathies may also becaused by the degeneration of specific nerve system cells, e.g. one ofmotor nerve degenerative diseases, amyotrophic lateral sclerosis, andone of neuro-degenerative diseases, Alzheimer's disease, which are motornerve degenerative diseases. The protein of the present invention can beused for the treatment of neuropathy caused by the nerve injury or nervedegeneration stated above. In addition, it can be administered topatients at peripheral nerve or artificial nerve grafting for nerveinjury. Furthermore, it is also useful for nervous function disorder dueto productive disorder.

The present invention further provides a method of producing the proteinof the present invention using an antibody column. This antibody columncan be obtained by binding an antibody cross-reacting with the proteinof the present invention to a supporting medium of column. In this case,the entire protein or its fragment having an antigen determinant can beused as an antigen. The antibody herein includes both monoclonal andpolyclonal antibodies and a chimeric antibody produced by the knownmethods, i.e., a “recombinant” antibody. Generally, polyclonal antibodyincludes various antibodies against various antigen determinants(epitopes), whereas monoclonal antibody is an antibody against a singleantigen determinant on an antigen.

Regarding polyclonal antibody, the antiserum can be obtained by aprocess of emulsifying the protein of the present invention to Freund'scomplete adjuvant, immunizing a rabbit, mouse, rat, guinea pig or sheep,boosting at intervals of two weeks using Freund's incomplete adjuvantand then bleeding. A specific antibody against the protein of thepresent invention can be obtained, if necessary, by carrying out anammonium sulfate fractionation for the obtained antiserum to roughlypurify IgG, and then absorbing the roughly purified product with anaffinity column to which a purified protein is bound (e.g. the use ofCNBr activating Sepharose).

An advantage of monoclonal antibodies is that these antibodies can besynthesized by hybridoma in a culture medium into which no otherimmunoglobulins are mixed. Monoclonal antibodies are prepared from theculture supernatant of hybridoma, or from mouse abdominal dropsy whichis induced by inoculating hybridoma into its abdominal cavity. Hybridomatechnique firstly described by Kohler and Milstein (Eur. J. Immunol. 6,511-519 (1976)) can broadly be used to generate hybrid cell systemshaving high-level monoclonal antibodies against a large number ofspecific antigens. Monoclonal antibodies can be prepared by thefollowing process: The protein of the present invention, along with anadjuvant such as a dead cell body, is injected into the abdominal cavityof mouse (e.g. BALB/c) to immunize it, and after a booster immunityconfirming the generation of antibody, and the spleen is extirpated fromthe mouse. After splenic cells are prepared, those cells are quicklyfused with a myelomain cell strain (e.g. X63, NS-1) in a HAT-medium(containing hypoxanthine, aminopterin and thymidine) in the presence ofpolyethylene glycol (e.g. #4000). After HAT selection of hybridoma andthe screening of a specific antibody-generating cell, this cell isinjected into a mouse's abdominal cavity and cloned to obtain amonoclonal antibody. The detailed method of preparing a monoclonalantibody is described, for example, in Tatsuo Iwasaki et al, “MonoclonalAntibodies—Hybridoma and ELISA” (1987) Kodansha Scientific, Tokyo,Japan.

The protein of the present invention can be separated and purified by aprocess of: preparing an antibody column by binding the above-obtainedantibodies to gels such as an agarose gel of Sepharose (Pharmacia)activated with cyanogen bromide; passing liquid derived from naturalsources (e.g. a conditioned medium having a nerve regeneration promotingactivity, or a human lung, kidney, placenta, etc.), recombinant cells orcultures through the column to absorb it; and eluting the protein of thepresent invention using salt concentration gradient, pH change and adenaturizing agent.

In order to stabilize the remedies containing the protein of the presentinvention, stabilizers such as saccharides and surfactants can be used.Examples of stabilizers include the following:

Saccharides used as a stabilizer include mannitol, lactose, sucrose,maltose, glucose, inositol, xylose, sorbitol, fructose, galactose,ribose, mannose, cellobiose, cyclodextrin, etc. Among these, sorbitol,mannitol and sucrose are preferable.

Surfactants used for the stabilizer include polyoxyethylene hydrogenatedcastor oil; polyoxyethylene castor oil; polyoxyethylene sorbitan fattyacid ester such as polysorbate 80 and polyoxyethylene sorbitanmonolaurate (sometimes called polysorbate 20); sorbitan fatty acid estersuch as polyoxyethylene polyoxypropylene glycol and sorbitan monooleate;sucrose fatty acid ester such as sucrose monolauric acid ester;benzethonium chloride; aromatic quaternary ammonium salt such asbenzalkonium chloride; sodium caprylate; sodium sulfite, etc. Amongthese, polysorbate 80, polysorbate 20 and polyoxyethylene hydrogenatedcastor oil are preferable.

In the remedies of the present invention, these saccharides can be usedwithin a range from 0.1 to 50% (w/v), and the surfactants within a rangefrom 0.0001 to 50% (w/v).

The remedies containing the protein of the present invention do not onlyinclude the above-stated various proteins having a neurotrophicactivity, but also the protein of the present invention which ischemically modified by binding to at least one water soluble polymer.The water soluble polymers are selected from e.g. polyethylene glycol,monomethoxy-polyethylene glycol, dextran, poly(N-vinyl-pyrrolidone)polyethylene glycol, propylene glycol homopolymer, polypropyleneoxido/ethylene oxide copolymer, polyvinyl alcohol, etc. These polymerscan form covalent bonds through an α-amino group at N-terminal ofprotein or a ε-amino group of lysine and a reaction group such asaldehyde. It is particularly preferable for the present invention that,by reacting the reactive PEG molecule with the protein of the presentinvention, reactive polyethylene glycol (PEG) is added thereto. Further,the molecular weight of PEG is preferably from 6 kDa to 50 kDa.

The protein of the present invention is useful as a nerve regenerationpromoting agent for neuropathy which promotes axonal regeneration andthe recovery of nerve tissues, and supports their survival, as describedabove and below.

EXAMPLES

The present invention will now be further described with examples, butnot limited thereto.

Regeneration of peripheral nerves was evaluated in vitro in thefollowing examples.

The dorsal root ganglion (DRG) with nerve stumps was excised from ananimal and then cultured in collagen gel. The cultured system was usedas an in vitro model. Using this system, assay for axonal regenerationand for neural survival activity by a factor were performed as describedpreviously (H. Horie et al., NeuroReport, 8, 1955-1959, 1997). DRGs (T2to T11) with nerve bundles, 1 to 2 mm in length, were excised from a3-month-old Wistar rat. The DRG was placed in a collagen solution [whichwas prepared by mixing (A) a solution of 0.5% collagen (type I)dissolved in a diluted acetic acid solution, (B) a 10-fold concentratedminimum essential medium (MEM), and (C) 100 ml of a solution containing2.2 g of NaHCO₃ and 4.77 g of HEPES dissolved in 0.05 NNaOH at a ratioof A:B:C=8:1:1 (by volume)] on a culture dish on ice. The dish washeated at once to 37° C., kept at 37° C. for 5 minutes, so that thecollagen solution was converted to a gel phase. Subsequently, the dishwas filled with Ham's F12 medium containing 5 μg/ml of insulin, 5 μg/mlof transferrin, 20 nM progesteron, 30 nM sodium selenite, and 0.1 mMputrescine, and then cultured at 37° C. in the air saturated with vaporcontaining 5% CO₂.

Nerve regeneration-promoting factors at various concentrations andfractionated fractions being subjected to purification were added to themedium and cultured for 6 to 7 days. The number of regenerating axonsfrom the transected nerve ends was measured under a phase contrastmicroscope. For each DRG, peripheral and central transected nerve endswere separately measured for the number of regenerating axons. Averageand standard error were calculated for each of all DRG measured, and thesignificance of the activity was statistically evaluated.

Example 1

Purification of mRNA from Rat Primary-Cultured Hepatocytes

Since Horie H. et al. found a nerve regeneration-promoting activity inthe supernatant of a rat hepatocyte primary culture (Neuroreport, 2,521-524, 1991), this primary-cultured cell was selected as a materialfor cDNA cloning of a rat nerve regeneration-promoting factor andsubjected to the following experiments.

Total RNA was extracted using ISOGEN [RNA extraction reagentmanufactured by NIPPON GENE; AGPC method (Chomczynski P, et al., Anal.Biochem. 162, 156-159, 1987)]. Rat hepatocytes were prepared by theenzyme perfusion method (Toshikazu Nakamura, Laboratory Procedures forHapatic Primary Cell Culture, 1987, Gakkai Shuppan Center, Tokyo,Japan), and cultured in a collagen-coated culture flask containing aserum free culture medium that had been prepared by adding 5 μg/ml ofinsulin (manufactured by SIGMA), 0.01 μg/ml of EFG (manufactured byTOYOBO CO., LTD., Japan), and 0.3 μg/ml of aprotinin (manufactured bySIGMA) to William's E medium. The primary cell culture prepared from theliver of a 8-week-old rat were inoculated at 8×10⁶ cells per flask, intotwenty five 175 cm² culture flasks (manufactured by Falcon), and thencultured in an incubator in 5% CO₂ gas at 37° C. for 2 days. Next theculture medium was removed from each flask, 4 ml of ISOGEN was added perflask followed by thorough suspension, thereby collecting the mixture.Using a 50 ml syringe with a 22G injection needle, suction and ejectionof the mixture was repeated about 20 times until the mixture almost lostits viscosity. 20 ml of chloroform was added and mixed with the mixture,followed by centrifugation at 12,000 G for 15 minutes. The supernatantwas carefully transferred into another tube, and 50 ml of isopropylalcohol was added to the tube and mixed, followed by centrifugation at12,000 G for 10 minutes. The resultant pellet was washed once with asmall quantity of 70% ethanol, thereby obtaining approximately 2 mg oftotal RNA from approximately 2×10⁸ hepatocytes.

Poly (A)⁺ RNA was purified from the total RNA using mRNA PurificationKit (manufactured by Pharmacia Corporation; method using Oligo dTcellulose). 90 μg of poly (A)⁺ RNA was obtained from approximately 2 mgof the total RNA.

Example 2

Construction of cDNA Library Derived from Rat Primary-CulturedHepatocytes for Expression Cloning

Double-stranded cDNA, having an EcoRI recognition site on the Met sideand a NotI recognition site on the Poly (A) side, was synthesized usingTimeSaver™ cDNA Synthesis Kit [manufactured by Pharmacia; a kit for cDNAsynthesis based on modified Gubler & Hoffman method (Gene 25, 263-269,1983)] and DIRECTIONAL CLONING TOOLBOX [manufactured by PharmaciaCorporation; a set containing a primer for cDNA synthesis having NotIsequence: 5′-AACTGGAAGAATTCGCGGCCGCAGGAA(T)₁₈-3′ (SEQ ID NO:11) andadaptors for addition of EcoRI sequence: 5′-AATTCGGCACGAGG-3′ (SEQ IDNO:12), and 5′-CCTCGTGCCG-3′] (SEQ ID NO: 63) from 5 μg of poly (A)⁺ RNAobtained in Example 1. Two third of the synthesized cDNA was linked toan 1.5 μg of the expression vector pEF18S that had previously beendigested with EcoRI and NotI (Ohashi H. et al., Proc. Natl. Acad. Sci.USA 91, 158-162, 1994). Next the product was divided into 12 equalparts, and each part was transformed into 1000 μl of Competent High E.coli DH5 (manufactured by TOYOBO CO., LTD., Japan). As a result, 12pools of cDNA library, each having 8.3×10⁴ transformants, were prepared(1.0×10⁶ transformants in total).

Example 3

Preparation of pRLF cDNA by Expression Cloning

Each pool of the cDNA library of rat primary-cultured hepatocyte asprepared in Example 2 was cultured overnight in 15 ml of 2×LB medium (2%Tryptone, 1% yeast extract, 1% NaCl, 0.2% Glucose) containing 50 μg/mlof ampicillin. 0.5 ml of pressure-sterilized glycerin was added andmixed with 0.5 ml of the cultured solution, and the mixture was storedat −80° C. 200 μl of the stored cell solution was cultured overnight in50 ml of LB medium (1% Tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1%Glucose) containing 50 μg/1 ml of Ampicillin. Subsequently plasmid DNAwas extracted basically as described in Molecular Cloning (Sambrook etal., Cold Spring Harbor Laboratory Press, 1989). The 50 μg out of thetotal 200 μg of the extracted plasmid DNA was transfected into COS1cells according to the slightly modified DEAE-dextran method includingchloroquine treatment (Sompayrac LM, et al., Proc. Natl. Acad. Sci. USA78, 7575-7578, 1981; Luthman H, et al., Nucl. Acids Res., 11, 1295-1308,1983) as shown below. 1.5×10⁶ COS1 cells (ATCC CRL1650) suspended inIscove's Modified Dulbecco's Medium (IMDM) containing 10% fetal calfserum (FCS) were inoculated onto plastic tissue culture flask(manufactured by CORNING) with 225 cm² culture surface area coated withcell matrix (manufactured by IWAKI GLASS CO., LTD., Japan), and thencultured overnight in an incubator with 5% CO₂ gas at 37° C. On theother hand, each pool of the plasmid DNA that had been dissolved in 250μl of HBS (21 mM HEPES—145 mM NaCl, pH 7.1) was mixed with 25 ml of IMDMcontaining 250 mg/ml of DEAE-dextran (manufactured by Pharmacia), 48 mMchloroquine (manufactured by SIGMA) and 8% (v/v) Nu-Serum (manufacturedby Collaborative). Immediately before transfection, the mixture wasadded to the above-described COS 1 cells that had been washed twice withIMDM. The mixture was cultured in an incubator with 5% CO₂ gas for 3hours at 37° C. Subsequently the culture supernatant was removed bysuction and the flask was washed twice with IMDM. 65 ml of IMDMcontaining 0.02% bovine serum albumin, 20 μg/ml of human insulin(manufactured by GIBCO BRL), 20 μg/ml of human transferrin (manufacturedby GIBCO BRL), 40 μM monoethanolamine (manufactured by SIGMA), and 0.1μM sodium selenite (manufactured by SIGMA) was added, and then the cellwas cultured in an incubator with 5% CO₂ gas for 3 days at 37° C.,followed by collection of a culture supernatant. The supernatant wasthoroughly dialyzed against a F12 medium, and then the nerveregeneration-promoting activity was measured in the above-mentioned invitro assay system. As a result, nerve regeneration-promoting activitywas identified in the culture supernatant of COS1 cells that had beentransfected with a pool of the plasmid DNA.

Next, the stored bacterial pool, in which the activity had been found,was diluted 1:10⁵ in CIRCLEGROW™ medium (manufactured by BIO 101)containing 50 μg/ml of Ampicillin. Then 2.5 ml each of the dilutedsolution was dispensed into 18 tubes, and then cultured overnight. [50μl of an aliquot was spread over LB agar medium (supplemented with 1.5%agar) containing Ampicillin, and then cultured overnight at 37° C. Bycounting the number of colonies appeared on the medium, it was confirmedthat one tube contained 8.2×10³ cDNA clones. That is, a pool containing8.2×10³ clones was generated.] 0.5 ml of pressure-sterilized glycerolwas added to 0.5 ml of the culture solution, and then stored at −80%.Plasmid DNA was purified from the remaining 2 ml as described above,thereby obtaining 5 μg of the plasmid DNA per pool. Further, the plasmidDNA was transfected into COS1 cells as described above. From now, theexperiment was scaled down using petri-dishes with 60 mm diameter. Nerveregeneration-promoting activity in the resulting culture supernatant wasmeasured, thereby obtaining one pool with the activity. The number ofcDNA clones contained in the pool with the activity was reduced from8.3×10⁴ to 8.2×10³, 1.5×10³, and 220 in order by repeating suchscreening. When two or more pools having the activity were obtained in asingle round of screening, one of these pools was used for the nextround of screening. When the number of clones of the pool reached 220,the stored bacterial pool was diluted, inoculated on LB agar mediumcontaining Ampicillin, and then cultured overnight for the sake offormation of colonies. 540 colonies were individually picked up from theformed colonies. 20 of these colonies were inoculated together in 2.5 mlof CIRCLEGROW™ medium (manufactured by BIO 101) containing 50 μg/ml ofAmpicillin, thereby obtaining 27 pools. Plasmid DNA was purified from 2ml of each overnight culture pool as described above and transfectedinto COS1 cells, and then the presence of a nerve regeneration-promotingactivity in the culture supernatant was measured. Thus, a pool havingthe activity was obtained. Plasmid DNA was purified from each of the 20colonies contained in the pool with the activity, as described above.Then the plasmid DNA was transfected into a COS1 cell, and the nerveregeneration-promoting activity in the culture supernatants wasmeasured. A cDNA clone having the activity was eventually obtained andnamed pRLF.

Example 4

Sequence Analysis of Rat pRLF cDNA

The plasmid DNA of the cDNA clone pRLF obtained in Example 3 waspurified basically as described in Molecular Cloning (Sambrook et al.,Cold Spring Harbor Laboratory Press, 1989). Approximately 700 μg ofplasmid DNA was obtained from 40 ml of the overnight culture of pRLFclone in CIRCLEGROW™ medium containing 50 μg/ml of ampicillin. Theobtained plasmid DNA was amplified by PCR using universal primers andoligonucleotide primers of approximately 20 bases that had beensynthesized based on the determined cDNA sequence, and using Taq DyeDeoxy™ Terminator Cycle Sequencing Kit (manufactured by Perkin-ElmerCorporation; dideoxy method using fluorescent dye and PCR: Sanger F. etal., Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977). [Theoligonucleotide primers of approximately 20 bases had been synthesizedusing a 394 DNA/RNA synthesizer (manufactured by Perkin-ElmerCorporation) which is based on the β-cyanoethyl amidite method.Purification of synthetic DNA was performed using an OPC column(manufactured by Perkin-Elmer Corporation). The OPC column is filledwith reversed phase silica gel and is used to purify synthetic DNAhaving trityl group. The purified synthetic DNA was dissolved in TEsolution to 20 μM and stored at −20° C. until use.] Then the cDNAnucleotide sequence was determined by a 373A DNA sequencer (fluorescentsequencer, manufactured by Applied Biosystems).

The nucleotide sequence was shown in the Sequence Listing (SEQ ID NO:2).

Example 5

Detection of pRLF mRNA in Various Rat Tissues

To determine if pRLF is a full-length clone of interest and which organproduces mRNA of interest, Northern blotting was performed using RatMultiple Tissue Northern Blot [manufactured by CLONTECH; nylon membraneto which poly (A)+ RNAs from different rat tissues were blotted].Northern blotting was performed basically as described in MolecularCloning (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).Prehybridization was conducted for 1 hour at 42° C. in 20 ml of asolution containing 50% formamide, 5×SSC, 5× Denhardt's solution, 1%SDS, and 200 μg/ml of salmon sperm DNA. Probes used herein were preparedby digesting pRLF with restriction enzymes NotI and EcoRI, separatingcDNA fragments of approximately 600 bp by electrophoresis using 0.8%agarose gel (manufactured by FMC BioProducts), purifying the fragmentsusing Prep-A-Gene DNA purification Kit (manufactured by Bio-RadLaboratories, Inc.), and labeling the products with ³²P using a RandomPrimer DNA labeling kit (manufactured by Takara Shuzo Co., Ltd., Japan;a kit based on the random primer method described in Anal. Biochem.,132, -13, 1983). Hybridization was conducted by adding the probes to 20ml of a solution with the same composition as used in prehybridizationand allowing the mixture to react for 20 hours at 42° C. The filter waswashed in 2×SSC/0.1% SDS solution for 5 minutes at room temperature, andwashed twice in 0.1×SSC/0.1. % SDS solution for 30 minutes at 68° C.,followed by analysis using a FUJIX bio-image analyzer BAS 2000(manufactured by Fuji Photo Film Co., Ltd., Japan). As a result, theexpression of mRNA was seen in the heart, brain, spleen, lung, liver,skeletal muscle, and kidney, with relatively high expression in the lungand liver (FIG. 1). Since the strand length of a band is about 1.6 Kb,pRLF was thought to be an incomplete length clone.

Example 6

Isolation of a Full-Length Clone of pRLF

The 5′ terminal was recovered by the 5′ RACE method because pRLF wasthought to be an incomplete length clone based on the results of Example5. First, two PCR primers were synthesized, which correspond to the mostupstream of the cDNA inserted in pRLF. The sequences are as follows:

-   Flf: 5′-GTGGTCAGGTTTGGCTCATA-3′ (complementary to the nucleotides    52-71 of SEQ ID NO:2 in the Sequence Listing; SEQ ID NO:13).-   Flg: 5′-TGCTCTTCACAGGCCCCCT-3′ (complementary to the nucleotides    33-51 of SEQ ID NO:2 in the Sequence Listing; SEQ ID NO:14).    Further, a primer for sequencing pEF18S was constructed. The    sequence is as follows.-   EF1α-2:5′-GGATCTTGGTTCATTCTCAAG-3′ (SEQ ID NO:15; located outside    EcoRI of a cloning site EcoRI-NotI of pEF18S, toward this cloning    site).

PCR was performed using 10 pmol each of the synthesized primers (Flf andEF1α-2) and using the plasmid of the cDNA library (independent clones;3.2×10⁶) prepared from the mRNA of rat primary-cultured hepatocytes as atemplate, in a similar manner to that of Examples 1 and 2. Using TaKaRaLA Taq (manufactured by Takara Shuzo Co., Ltd.), PCR was performed in avolume of 100 μl using a GeneAmp™ PCR System 2400 (manufactured byPerkin-Elmer Corporation). The PCR reaction cycle was repeated 35 timesafter denaturation for 5 minutes at 94° C., each cycle consisting ofdenaturation for 30 seconds at 94° C., annealing for 30 seconds at 57°C., and synthesis for 2 minutes at 72° C.; followed by synthesis forfurther 5 minutes at 72° C. Further, PCR was performed under the sameconditions as described above using 10 pmol each of the synthesizedprimers, Flg and EF1α-2, and using 1 μl of the reaction solution diluted4-fold as a template. The reaction solution was subjected toelectrophoresis using 2% agarose gel. Thus a relatively large and clearband was recovered and the product was purified therefrom usingPrep-A-Gene DNA Purification Kit. The nucleotide sequence of thisfragment was analyzed directly by a type 377 DNA sequencer (manufacturedby Perkin-Elmer Corporation) using the synthesized primers, Flg andEf1α-2, and using a Taq Dye Deoxy™ Terminator Cycle Sequencing FS Kit(manufactured by Perkin-Elmer Corporation). Thus an upstream sequence of332 bases was obtained (SEQ ID NO: 3). However, since it was still shortof the analytical result of Northern blotting (total length ofapproximately 1.6 Kb), the following 5′ RACE was further repeated.First, two PCR primers were synthesized, which correspond to the Nterminal side of the obtained upstream sequence. The sequences are asfollows:

-   Flh: 5′-CCAAGTCCGTATCTCCATCA-3′ (complementary to the nucleotides    118-137 of SEQ ID NO: 3 in the Sequence Listing; SEQ ID NO:16).-   Fli: 5′-GGCAGTCCAGTATGCTACAT-3′ (complementary to the nucleotides    36-55 of SEQ ID NO: 3 in the Sequence Listing; SEQ D NO: 17).    PCR was performed using 10 pmol each of the synthesized primer, Flh,    and Anchor Primer (attached to 5′-RACE-Ready cDNA; corresponding to    an anchor added to the 5′ end of the cDNA) and using rat spleen    5′-RACE-Ready cDNA (manufactured by CLONTECH) as a template. Using    TaKaRa LA Taq (manufactured by Takara Shuzo Co., Ltd. Japan), PCR    was performed in a volume of 50 μl using GeneAmp™ PCR System 2400    (manufactured by Perkin-Elmer Corporation). The PCR reaction cycle    was repeated 30 times, each consisting of denaturation for 45    seconds at 94° C., annealing for 45 seconds at 57° C., and synthesis    for 2 minutes at 72° C.; followed by synthesis for further 5 minutes    at 72° C. Furthermore, PCR was performed under the same conditions    employed above in a volume of 100 μl using 20 pmol each of the    synthesized primer, Fli, and Anchor Primer, and using 4 μl of the    reaction solution 10-fold diluted as a template. The reaction    solution was subjected to electrophoresis using 2% agarose gel. Thus    the clearest band was removed and the product was purified therefrom    using Prep-A-Gene DNA Purification Kit. This fragment was cloned    into a PCR™II vector (TA Cloning™ Kit; manufactured by Invitrogen).    PCR was performed using the resultant colonies as templates, and    using 7 pmol each of M13 Reverse Primer (5′-CAGGAAACAGCTATGAC-3′;    SEQ ID NO:18), M13 (−20) Forward Primer (5′-GTAAAACGACGGCCAGTG-3′;    SEQ ID NO:19). That is, the PCR was performed in a volume of 30 μl    by GeneAmp™ PCR System 2400 (manufactured by Perkin-Elmer    Corporation) using AmpliTaq™ (manufactured by Perkin-Elmer    Corporation). The reaction cycle was repeated 35 times after    denaturation for 5 minutes at 94° C., each cycle consisting of    denaturation for 30 seconds at 94° C., annealing for 30 seconds at    47° C., and synthesis for 1 minute at 72° C.; followed by synthesis    for further 5 minutes at 72° C. The reaction solution was subjected    to electrophoresis using 2% agarose gel. Thus a band was recovered    and the product was purified therefrom using Prep-A-Gene DNA    Purification Kit. The nucleotide sequence of this fragment was    analyzed by a type 377 DNA sequencer (manufactured by Perkin-Elmer    Corporation) using a Taq Dye Deoxy™ Terminator Cycle Sequencing FS    Kit (manufactured by Perkin-Elmer Corporation) and using Anchor    Primer and Fli. Therefore, a more upstream sequence of 335 bases was    obtained (SEQ ID NO:4). At this time, a PCR primer was synthesized,    which corresponds to the N terminal of this upstream sequence. The    sequence is as follows:-   Flj: 5′-TCCTCCTCGACACGCACTCC-3′ (complementary to the nucleotides    64-83 of SEQ ID NO:4 in the Sequence Listing; SEQ ID NO:20).    PCR was performed using 20 pmol each of the synthesized primer, Flj,    and Anchor Primer, and using 4 μl of the above PCR reaction solution    10-fold diluted, for which rat spleen 5′-RACE-Ready cDNA as a    template, and Flh and Anchor Primer had been used. Using TaKaRa LA    Taq (manufactured by Takara Shuzo Co., Ltd. Japan), PCR was    performed in a volume of 100 μl using a GeneAmp™ PCR System 2400    (manufactured by Perkin-Elmer Corporation). The PCR reaction cycle    was repeated 30 times, each consisting of denaturation for 30    seconds at 94° C., annealing for 30 seconds at 63° C., and synthesis    for 1.5 minutes at 72° C.; followed by synthesis for 5 minutes at    72° C. The reaction solution was subjected to electrophoresis using    2% agarose gel. Thus the clearest and longest band was recovered and    the product was purified therefrom using Prep-A-Gene DNA    Purification Kit. This fragment was cloned into a PCR™II vector.    Then in the same manner as described above, the fragment was    prepared from the colony by PCR, thereby determining its nucleotide    sequence. The Primers used herein were M13 Reverse Primer as    described above and T7 Primer (5′-TAATACGACTCACTATAGGG-3′; SEQ ID    NO: 21). Thus, another upstream sequence of 317 bases (SEQ ID NO:5)    was obtained and its full length was 1571 bases (SEQ ID NO:6). Since    this full length was consistent with the analytical results of    Northern blotting, it was concluded that a complete length clone was    obtained.

Homology search was conducted using DNA analysis software “DNASIS”(manufactured by HITACHI SOFTWARE ENGINEERING CO., LTD.). It was shownthat the antisense sequence of SEQ ID NO: 6 shared a homology of 85.4%with human Bcl-2 binding component 3 (GENBANK ACCESSION NO. U82987) atnucleotide level. On the other hand, analysis using the same softwareshowed that only ORF of maximum 24 amino acids was present in the clonepRLF (SEQ ID NO:2) that had been obtained by expression cloning.Accordingly, it was thought that protein (peptide) itself encoded by theclone pRLF does not possess nerve regeneration activity, but a substancewith nerve regeneration activity may be secreted from COS1 cells by somemechanisms due to introduction of the clone pRLF. To prove thehypothesis, the following experiment was conducted.

Example 7

Purification of Nerve Regeneration-Promoting Factor from CultureSupernatant of pRLF-Transfected COS1 Cells

Preparation of Culture Supernatant of pRLF-Transfected COS1 Cells

Approximately 300 L of the culture supernatant of pRLF-transfected COS1cells were prepared to purify a nerve regeneration-promoting factor thatis secreted in the culture supernatant of the COS1 cells. Thispreparation is as described below. First, pRLF clone was shake-culturedin LB medium containing 50 μg/ml of Ampicillin overnight at 37° C. Here,1.5 L each of this medium was put into a 3L Sakaguchi's flask. Then 170L of the culture was centrifuged to obtain E. coli (wet weight 850 g).Using a plasmid extraction kit (RPM™-4G, manufactured by BIO 101), pRLFplasmid was extracted from 5 g of the E. coli per batch. Thus 300 mg ofpRLF plasmid was obtained from 170 L of the overnight culture solution.

Transfection of the plasmid pRLF into COS1 cells was performed asdescribed in Example 3. Thus total 294 L of the culture supernatant ofpRLF-transfected COS1 cells was obtained and used as a source forpurification.

Purification of Nerve Regeneration-Promoting Factor from the CultureSupernatant of pRLF-Transfected COS1 Cell

The obtained supernatant was divided into 7 lots, 30 to 50 L each, thenpurified, because 294 L of the obtained supernatant (total amount ofprotein; 81678 mg) could not be treated at once. Of these lots, atypical purification is explained for a certain lot. For allpurification steps, nerve regeneration-promoting activity was measuredusing the above-described DRG assay system. Protein was quantified byCoomasie dye binding method (with the reagent manufactured by PIERCE)for steps (1) to (3) and based on absorbance at 280 nm for (4) andfollowing steps. Lots are summarized as follows:

-   Lot 1: 42.81 L, Protein concentration 0.297 mg/ml Total amount of    protein 12698 mg;-   Lot 2: 44.73 L, Protein concentration 0.241 mg/ml, Total amount of    protein 10760 mg;-   Lot 3: 34.16 L, Protein concentration 0.301 mg/ml, Total amount of    protein 10290 mg;-   Lot 4: 43.05 L, Protein concentration 0.266 mg/ml, Total amount of    protein 11438 mg;-   Lot 5: 46.30 L, Protein concentration 0.261 mg/mil, Total amount of    protein 12070 mg;-   Lot 6: 43.92 L, Protein concentration 0.290 mg/mil, Total amount of    protein 12752 mg;-   Lot 7: 39.15 L, Protein concentration 0.298 mg/ml, Total amount of    protein 11670 mg.    (1) Fractionation with Ultrafiltration Membrane

First, Lot 1, the culture supernatant of pRLF-transfected COS1 cells(42.81 L, Protein concentration; 0.297 mg/ml, total amount of protein;12,698 mg) was centrifuged at 8000 RPM for 30 minutes to remove deadcell debris, thereby collecting the supernatant. Next, the resultingsupernatant was filtered through a 100 kDa cutoff ultrafiltrationmembrane (manufactured by PAUL FILTRON, membrane area 0.46 m²) followedby concentration using a 5 KDa cut-off ultra filtration membrane(manufactured by PAUL FILTRON, membrane area 0.46 m²). The threeobtained fractions were a fraction of 5 KDa or less (40.73 L, proteinconcentration: detection limit or less), a fraction of 5 KDa or more and100 KDa or less (320 ml, protein concentration: 39.252 mg/ml, totalamount of protein: 12560 mg), and a fraction of 100 KDa or more (800 ml,protein concentration: 3.084 mg/ml, total amount of protein: 2467 mg).The three fractions were subjected to DRG assay. Since nerveregeneration-promoting activity was detected in the fraction of 5 KDa ormore and 100 KDa or less, this fraction was used for the next step.

(2) TSKgel QAE-TOYOPEARL 550C (Strong Anion Exchange Chromatography)

The fraction of 5 KDa or more and 100 KDa or less obtained in (1) (320ml, protein concentration: 39.252 mg/ml, total amount of protein: 12560mg) was diluted 4-fold using 20 mM Tris-HCl buffer (pH 8.0).Subsequently, the diluted solution was loaded at a flow rate of 15ml/min to a TSKgel QAE-TOYOPEARL 550C column (manufactured by TOSOHCORPORATION, Japan; φ5 cm×10 cm) equilibrated with 20 mM Tris-HCl buffer(pH 8.0) at 4° C. The flow-through fraction was eluted (1900 ml, proteinconcentration: 0.106 mg/ml, total amount of protein: 201 mg). Next, theeluate was replaced by 20 mM Tris-HCl buffer (pH 8.0) with 750 mM NaCland loaded to the column at a flow rate of 5 ml/min, so that an adsorbedfraction Q2 was eluted (590 ml, protein concentration: 20.631 mg/ml,total amount of protein: 12172 mg). As a result of DRG assay, nerveregeneration-promoting activity was detected in the adsorbed fractionQ2. Hence, the adsorbed fraction Q2 was used for the next step.

(3) Sephacryl S-200 HR (Gel Filtration Chromatography)

TSKgel QAE-TOYOPEARL 550C column adsorbed fraction Q2 obtained in (2)(590 ml, protein concentration 20.631 mg/ml, total amount of protein12172 mg) was concentrated to 100 ml using an ultrafiltration unit(manufactured by Amicon; YM3 membrane, 76 mm in diameter). Then, 50 mlof the concentrate 100 ml was loaded at a flow rate of 2.5 mL/min at 4°C. to a Sephacryl S-200 HR column (manufactured by Amersham pharmaciabiotech; φ5 cm×100 cm) that had been previously equilibrated with PBS.50 ml each of the eluate was collected in a tube, and subjected to DRGassay. As a result, nerve regeneration-promoting activity was detectedin Fr10 to Fr16, corresponding to the molecular weight ranging from 30KDa to 5 KDa (350 ml, protein concentration: 4.303 mg/ml, total amountof protein: 1506 mg). Similarly, the remaining 50 ml of concentrate wasalso fractionated using a Sephacryl S-200 HR column, thereby obtainingactive fractions, Fr10 to Fr16 (350 ml, protein concentration: 4.480mg/ml, total amount of protein: 1568 mg). Sephacryl S-200 HR activefractions thus obtained by two rounds of separate chromatographies werecombined (700 ml, protein concentration 4.391 mg/ml, total amount ofprotein: 3074 mg) and then concentrated to 50 ml using a ultrafiltrationunit (manufactured by Amicon; YM3 membrane, 76 mm in diameter). Then,the concentrate was loaded again to a Sephacryl S-200 HR column(manufactured by Pharmacia Biotech, φ5 cm×100 cm) at a flow rate of 2.5ml/min at 4° C. The eluate was fractionated into 50 ml each, andsubjected to DRG assay. As a result, nerve regeneration-promotingactivity was detected in Fr13 and 14 corresponding to a molecular weightof 15 Kda (100 ml, protein concentration: 0.224 mg/ml, total amount ofprotein: 22.039 mg). Thus this fraction was used for the next step.

In this manner, purification steps of (1) to (3) above were conductedfor 7 lots, thereby obtaining Sephacryl S-200 HR active fractions foreach of these lots. Sephacryl S-200 HR active fractions for each lot aresummarized as follows:

-   Lot 1: 100 ml, Protein concentration 0.224 mg/ml, Total amount of    protein 22.039 mg;-   Lot 2: 50 ml, Protein concentration 0.313 mg/ml, Total amount of    protein 15.672 mg;-   Lot 3: 50 ml, Protein concentration 0.366 mg/ml, Total amount of    protein 18.300 mg;-   Lot 4: 50 ml, Protein concentration 0.316 mg/ml, Total amount of    protein 15.800 mg;-   Lot 5: 100 ml, Protein concentration 0.230 mg/ml, Total amount of    protein 22.950 mg;-   Lot 6: 50 ml, Protein concentration 0.305 mg/ml, Total amount of    protein 15.240 mg;-   Lot 7: 50 ml, Protein concentration 0.245 mg/ml, Total amount of    protein 12.250 mg.    (4) Shodex IEC DEAE-2025 (Weak Anion Exchange Chromatography)

Lot 1, Sephacryl S-200 HR active fraction (100 ml, proteinconcentration: 0.224 mg/ml, total amount of protein: 22.039 mg) and Lot2, Sephacryl S-200 HR active fraction (50 ml, protein concentration:0.313 mg/ml, total amount of protein: 15.672 mg) as obtained in (3),were combined (150 ml, protein concentration: 0.251 mg/ml, total amountof protein: 37.711 mg). Then this fraction was concentrated to 5 mlusing an ultra filtration unit (manufactured by Amicon; membrane YM3,diameter 76 mm). Subsequently 50 ml of 20 mM Tris-HCl buffer (pH 8.0)was added to the concentrate followed by re-concentration to 5 ml.Another round of this procedure was performed to exchange the bufferwith 20 mM Tris-HCl buffer (pH 8.0), thereby obtaining a Sephacryl S-200HR active fraction (12 ml, protein level: 3.143 mg/ml, total amount ofprotein: 37.711 mg). Using 20 mM Tris-HCl buffer (pH 8.0) as an eluentA, and 20 mM Tris-HCl buffer (pH 8.0) containing 750 mM NaCl as aneluent B, the fraction was loaded at a flow rate of 2 ml/min to a ShodexEC DEAE-2025 column (manufactured by Showa Denko K.K., Japan; φ2 cm×15cm) equilibrated with 0% B, at room temperature. After loading, thecolumn was eluted with 20% B in 10 minutes, followed by a 70 minuteslinear gradient from 20% B to 60% B. The eluate was fractionated into 4ml each and then subjected to DRG assay. As a result, nerveregeneration-promoting activity was detected in Fr 39 and 40 having NaCllevel of approximately 250 mM (8 ml, protein concentration: 0.02 mg/ml,total amount of protein: 0.16 mg). Thus this fraction was used for thenext step.

The remaining lots were purified in the same manner as above, so thatShodex EEC DEAE-2025 active fractions were obtained. That is, the lotswere divided into three groups, the combined fraction of Lots 3 and 4,the combined fraction of Lots 5 and 6, and the fraction of Lot 7. Thenthe groups were separately purified by Shodex EC DEAE-2025, therebyobtaining Shodex IEC DEAE-2025 active fractions.

The Shodex IEC DEAE-2025 active fractions for each of the group were assummarized below:

-   Lot 1 & 2: 8 ml, protein concentration: 0.02 mg/ml, total amount of    protein: 0.16 mg;-   Lot 3 & 4: 8 ml, protein concentration: 0.02 mg/ml, total amount of    protein: 0.16 mg;-   Lot 5 & 6: 8 ml, protein concentration: 0.025 mg/ml, total amount of    protein: 0.2 mg;-   Lot 7: 8 ml, protein concentration: 0.01 mg/ml, total amount of    protein: 0.08 mg.    (5) YMC-Pack Protein RP (Reverse Phase Chromatography)

The Shodex IEC DEAE-2025 active fractions of each group of lots asobtained in (4) were combined (32 ml, protein level: 0.0188 mg/ml, totalamount of protein 0.6 mg). Then the combined fraction was concentratedto 4 ml using a ultrafiltration unit (manufactured by Amicon; membraneYM 3, diameter 43 mm). 0.5 ml of 2-propanol, 0.5 ml of acetonitrile, and0.0025 ml of trifluoroacetic acid (TFA) were added to the concentrate sothat the concentrate became a final volume of 5.0025 ml, final organicsolvent concentration of 20%, and 0.05% of TFA concentration. Thismixture was loaded at a flow rate of 0.2 ml/min to a YMC-Pack PROTEIN RPcolumn (manufactured by YMC, φ2.1 mm×150 mm) equilibrated with 40% B,using 2-propanol/acetonitrile 1/1 (volume ratio) that contained 0.1% TFAas an eluent A and 0.08% TFA as an eluent B. After loading, the columnwas developed with a 30 minutes linear gradient from 40% B to 60% B. Theeluate was fractionated into 0.4 ml each, and then subjected to DRGassay. Thus nerve regeneration-promoting activity was detected in Fr 44and 45 having an organic solvent concentration of approximately 50% (0.8ml, protein level: 0.012 mg/ml, total amount of protein: 0.0096 mg).This fraction was used for the next step.

(6) Identification of Active Protein Band on the Electrophoretic Gel

Part of YMC-Pack PROTEIN RP active fractions as obtained in (5) wasanalyzed by electrophoresis. Electrophoresis was performed according tostandard techniques (Laemmli, Nature, vol. 227, 680-685, 1970). 5.4 μlout of 0.8 ml of the active fractions was subjected to centrifugalevaporation, followed by the addition of 10 μl of sodium dodecyl sulfate(SDS) gel electrophoresis sample buffer with no reducing agent. Then theproduct was treated for 5 minutes at 95° C., subjected to SDS gelelectrophoresis using 15-25% SDS-polyacrylamide precast gel(manufactured by Daiichi Pure Chemicals, Japan), and stained with2D-silver staining reagent (Daiichi silver staining kit; manufactured byDaiichi Pure Chemicals, Japan). Daiichi III low molecular weight marker(manufactured by Daiichi Pure Chemicals, Japan) was used as a molecularweight marker. As a result, multiple bands were detected on theelectrophoretic gel and the presence of multiple proteins in the activefractions was confirmed. To identify the active protein bands on theelectrophoretic gel, an experiment was conducted to extract nerveregeneration-promoting activity from the electrophoretic gel. 8 μl outof 0.8 ml of the active fractions was subjected to centrifugalevaporation, followed by the addition of 10 μl of SDS gelelectrophoresis sample buffer with no reducer. Then the product wastreated for 10 minutes at 55° C., subjected to SDS gel electrophoresisusing 15-25% SDS-polyacrylamide precast gel (manufactured by DaiichiPure Chemicals, Japan). A pre-stained broad range marker (manufacturedby New England Biolabs. Inc.) was used as a molecular weight marker.After electrophoresis, using a molecular weight of 16.5 KDa band of thepre-stained broad range marker as an index, a molecular weight regionlower than 16.5 KDa band out of the regions to which samples had beenadded was cut into 14 rows of gel each having 1.6 mm width. The cut 14rows of gel were separately added into an Eppendorf tube each containing500 μl of purified water with 1.25 μg of transferrin, and then the tubeswere set in a rotator and rotated at 4° C. After 16 hours, the extractswere collected. Then another 500 μl of purified water containing 1.25 μgof transferrin was added to the tube. After rotating for 4 hours at 4°C., the first and second extracts were mixed together to form a total of1 ml of protein extract present in the gel. Subsequently, 20 μl of 1Mpotassium phosphate (pH 6.8) was added to each of the tubes to eliminatefree SDS present in the extract, and then the mixture was allowed, tostand for 4 hours at 4° C. Further, the mixture was centrifuged at10,000 rpm for 10 minutes to remove the precipitate, and the supernatantwas collected, thereby obtaining protein extract present in the gel.After the extract was dialyzed against an assay medium, and subjected toDRG assay, nerve regeneration-promoting activity was detected in No.3and No. 4 gel extracts corresponding to molecular weight ofapproximately 14500 Da. Thus it was found that the protein withmolecular weight of approximately 14500 Da was an active protein.

(7) Separation by Electrophoresis and Electroblotting ontoPolyvinylidene Difluoride (PVDF) Membrane

The remainder of YMC-Pack PROTEIN RP active fractions were all separatedby electrophoresis, because it was found that the active protein inYMC-Pack PROTEIN RP active fractions can be separated from otherproteins by electrophoresis as described in (6). 786.6 μl of YMC-PackPROTEIN RP active fraction was subjected to centrifugal evaporation,followed by addition of 30 μl of SDS gel electrophoresis sample bufferwith no reducing agent. The product was treated for 5 minutes at 95° C.,and subjected to SDS gel electrophoresis using 15-25% SDS-polyacrylamideprecast gel (manufactured by Daiichi Pure Chemicals, Japan). Daiichi IIIlow molecular weight marker (manufactured by Daiichi Pure Chemicals,Japan) and a pre-stained broad range marker (manufactured by New EnglandBiolabs, Inc.) were used. After electrophoresis, using a semi drytransfer system (manufactured by Owl, Inc.), the product was transferredto PVDF membrane (manufactured by Perkin-Elmer Corporation; ProBlott) ata constant current of 150 mA for 3 hours. An anolyte consisting of 0.3 MTris and 20% methanol (pH 10.4), a transfer membrane solution of 25 mMTris and 20% methanol (pH 10.4), and a catholyte of 25 mM Tris and 40 mMaminocaproic acid and 20% methanol (pH 10.4) were used. The transferredmembrane was stained with Coomassie brilliant blue (CBB) solutioncontaining 0.1% CBB R-250 in 40% methanol and 1% acetic acid. When thestained product was destained with 50% methanol, a staining patternequivalent to that of silver staining as described in (6) was detected.Moreover, a protein band with molecular weight of approximately 14500Da, the activity itself, was also stained. Therefore, purification ofnerve regeneration-promoting factor in the culture supernatant ofpRLF-transfected COS 1 cell was completed. The obtained activity, amountof protein was assumed to be approximately 200 ng according to thedegree of CBB staining (FIG. 2).

Example 8

Analysis of Partial Amino Acid Sequence of Nerve Regeneration-PromotingFactor

The amino acid sequence of the protein having nerveregeneration-promoting activity with a molecular weight of approximately14500 Da that had been transferred to a PVDF membrane as obtained inExample 7 (7) was analyzed according to Iwamatsu's method (Iwamatsu etal., New Basic Biochemical Laboratory Procedures, Vol. 4, 33-84,Maruzen, Tokyo, Japan; Akihiro Iwamatsu, Seikagaku, Vol. 63, No. 2,139-143, 1991, Japan; and Akihiro Iwamatsu, Electrophoresis, Vol. 13,142-147, 1992).

(1) Reduction and S-carboxy Methylation

The CBB-stained protein band on a PVDF membrane, having nerveregeneration-promoting activity with a molecular weight of approximately14500 Da, was cut out. Then the protein band was immersed in anEppendorf tube with 100 PI of a reduction solution containing 1 mg ofdithiothreitol (DI) [0.5 M Tris-HCl buffer (pH 8.3) containing 8Mguanidine, 0.3% ethylenediaminetetraacetic acid (EDTA), and 0.125 Mlysine] and allowed to stand for 1 hour at 60° C. Next 3 mg ofmonoiodoacetic acid was added to the solution, and then the solution wasvigorously stirred for 15 minutes while shading the tube. Afterstirring, the PVDF membrane was washed in order with 2% acetonitrile and0.1% SDS to remove excess reagent remaining on the membrane. Thus, theprotein having nerve regeneration-promoting activity with a molecularweight of approximately 14500 Da, and reduced and S-carboxymethylated onthe PVDF membrane, was obtained.

(2) Peptide Fragmentation, Peptide Mapping, and Amino Acid SequenceAnalysis

To obtain multiple information on internal amino acid sequences from theresultant protein, the protein was fragmented into peptides by enzymaticdigestion. Prior to this enzymatic digestion, PVDF membrane was immersedin 100 μl of 100 mM acetic acid containing 0.5% polyvinylpyrolidone(PVP)-40 and 1 mg of methionine, and then allowed to stand for 30minutes at room temperature. Thus the unbound portion of protein on themembrane was blocked. After blocking, the membrane was washed with 10%acetonitrile to remove excess reagents. The washed membrane wastransferred into a tube containing 20 mM Tris-HCl buffer (pH 9.0) with10% acetonitrile, followed by addition of 5 pmol of lysyl endopeptidase(Achromobacter Protease I; manufactured by WAKO PURE CHEMICALINDUSTRIES., LTD., Japan), allowing digestion to occur for 3 hours at40° C. Thus peptide fragments digested by enzymes and liberated from thePVDF membrane were collected in a digestion buffer. The peptidefragments present in the digestion buffer were loaded to a Symmetry C18reversed phase column (manufactured by Waters; φ 1.0 mm×50 mm) at a flowrate of 0.1 ml/min at 40° C. using 0.05% TFA as solvent A andisoparopanol:acetonitrile=7:3 with 0.02% TFA as solvent B, repectively.The column was eluted with a 30 minutes linear gradient from 1% B to 50%B, thereby obtaining fractions (FIG. 3). Each of the resultant peptidefragments was subjected to Edman degradation using a gas phase aminoacid sequencer (manufactured by Shimadzu Corp., PPSQ-2). The N-terminalPTH amino acids recovered in sequence were identified by C18 reversephase column chromatography based on the isocractic elution method. Theamino acid sequences of the fragments that could be identified aresummarized as follows (wherein amino acids are shown by one-letternotation):

-   AP2: PGECLRVRGEVA (SEQ ID NO:22);-   AP4: LPDGYE (SEQ ID NO:23);-   AP6: DSNNLCLHFN (SEQ ID NO:24).

A database was searched for these amino acid sequences. These sequenceswere identical to an amino acid sequence of human galectin-1 (AccessionNO. P09382; SWISS-PROT database). Hence, it was concluded that the nerveregeneration-promoting factor in the culture supernatant ofpRLF-transfected COS1 cells was the most likely to be monkey galectin-1.

Example 9

Cloning of Human Galectin-1 cDNA

First, PCR primers were synthesized based on GENBANK ACCESSION NO.J04456, corresponding to human galectin-1 cDNA. The sequences are asfollows:

-   HLEG1: 5′-TGCGCCTGCCCGGGAACATC-3′ (GENBANK ACCESSION NO. J04456,    15-34; SEQ ID NO:25);-   HLEG2: 5′-GAACATCCTCCTGGACTCAA-3′ (GENBANK ACCESSION NO. J04456,    2847; SEQ ID NO:26);-   HLEG6: 5′-GCTGCCTTTATTGGGGGCCA-3′ (the complementary strand to    GENBANK ACCESSION NO. J04456, 472-491; SEQ ID NO:27);-   HLEG8: 5′-GAGAGAGCGGCCGCATTGGGGGCCATGGGCTGGC-3′ (NotI site was added    to 5′ of the strand complementary to GENBANK ACCESSION NO. J04456,    463482; SEQ ID NO:28).

PCR was performed using 40 pmol each of the synthesized primers, HLEG1and HLEG6, and using 2 μl of E. coli stock of SuperScript™ Human LivercDNA Library (manufactured by GIBCO BRL) as a template. Using TaKaRa LATaq (manufactured by Takara Shuzo Co., Ltd. Japan), PCR was performed ina volume of 100 μl using GeneAmp™ PCR System 2400 (manufactured byPerkin-Elmer Corporation). The PCR reaction cycle was repeated 35 timesafter denaturation for 5 minutes at 94° C., each cycle consisting ofdenaturation for 30 seconds at 94° C., annealing for 30 seconds at 60°C., and synthesis for 1 minute at 72° C.; followed by synthesis forfurther 5 minutes at 72° C. Further, PCR was performed using 40 pmoleach of the synthesized primers, HLEG2 and HLEG8, and using 1 μl of thereaction solution under the same conditions except that annealingtemperature was 55° C. The reaction solution was treated withphenol-chloroform (1:1), added with 3M NaOAc ({fraction (1/10)} of thetotal volume) and ethanol (twice the total volume), and thencentrifuged, thereby obtaining pellet. The pellet was blunt-ended withT4 DNA polymerase (manufactured by Boehringer Manheim), digested withNotI, subjected to electrophoresis with 2% agarose gel, therebyrecovering a fragment of approximately 460 bp as predicted. The fragmentwas purified using Prep-A-Gene DNA Purification Kit. Further thefragments was digested with EcoRI, blunt-ended with T4 DNA polymerase,then linked to pEF18S that had been digested with NotI. E. coli DH5 wasused as a host cell. The insertion sequence of the obtained clone wasamplified by PCR (wherein the reaction cycle was repeated 35 times after5 minutes at 94° C., each cycle consisting of 30 seconds at 94° C./30seconds at 50° C./1 minute at 72° C.) using 7 pmol each of the primersEF1α-1 (located outside EcoRI of the cloning site EcoRI-NotI of pEP18S,toward this cloning site; 5′-CCTCAGACAGTGGTTCAAAG-3′; SEQ ID NO: 29),and polyAC2 (located outside of NotI of the cloning site EcoRI-NotI ofpEF18S, toward this cloning site; 5′-TGCATTCATTTTATGTTTCAG-3′; SEQ IDNO:30). Then the product was subjected to electrophoresis with 2%agarose gel. Thus the recovered fragment of approximately 500 bp aspredicted was purified using Prep-A-Gene DNA Purification Kit. Thenucleotide sequence of this fragment was analyzed by type377 DNAsequencer (manufactured by Perkin-Elmer Corporation) using primers,EF1α-1 and polyAC2, and using Taq Dye Deoxy™ Terminator Cycle SequencingFS Kit (manufactured by Perkin-Elmer Corporation). Thus, a clone pEFGal1(GENBANK ACCESSION NO. J04456, 50457) having the correct nucleotidesequence of human galectin-1 cDNA, from the initiation codon to thetermination codon, was obtained.

Example 10

Confirmation of DRG Activity of COS1 Cell Expression Protein of HumanGalectin-1 cDNA Clone pREFGal1 (referred to as COS1 expression Gal1(1-134))

The plasmid pEFGal1 with human galectin-1 cDNA incorporated therein asobtained in Example 9 was transfected to a COS1 cell. Then the presenceor absence of a nerve regeneration-promoting activity in Gal1 (1-134)that had been extracted from the COS1 culture supernatant and the COS1cells were confirmed.

The clone pEFGal1 was cultured overnight in 100 ml of LB mediumcontaining 50 μg/ml of Ampicillin and then subjected to centrifugation,thereby obtaining cells. Plasmid DNA was extracted from the resultantcells using QIAGEN Plasmid Maxi Kit (manufactured by QIAGEN). Theplasmid pEFGal1 was transfected to COS1 cells using Transfectum reagent[manufactured by Promega KK; dioctadecylamidoglycylspermin (DOGS)].5×10⁶ COS1 cells were inoculated on a plastic tissue culture flask withsurface area 225 cm², and cultured overnight in an IMDM mediumcontaining 10% FBS. After washing the culture cells with IMDM medium,6.5 ml of IMDM medium was added. Further, 6.5 ml of IMDM mediumcontaining 65 μg of the plasmid pEFGal1 and 6.5 ml of that containing325 μg of a Transfectum reagent were mixed together and then added tothe medium followed by culturing for 6 hours at 37° C. Next the culturesolution was removed by suction, and. 52 ml of an IMDM medium containing10% FBS was added to the flask, followed by 4 culturing for 2 days.After culturing, 50 ml of the culture supernatant and the cells werecollected. The culture supernatant was dialyzed against 2 L of PBScontaining 5 mM DTT overnight and then filtered. The collected cellswere homogenized in 10 ml of PBS containing 100 mM lactose and 5 mM DTT.Then the product was centrifuged at 10000 G for 30 minutes at 4° C.; andthe supernatant was collected, dialyzed overnight against 2 L of PBScontaining 5 mM DTT, and filtered, thereby obtaining the cell extract.The resulting culture supernatant and cell extract were separatelyloaded at a flow rate of 0.25 ml/min to a lactose agarose column(manufactured by HONEN CORPORATION; φ5.0 mm×50 mm) that had beenequilibrated with PBS containing 5 mM DTT, so that fractions that passedthrough the column were collected. Next, the eluting buffer was replacedby PBS containing 100 mM lactose and 5 mM DTT and adsorbed fractionswere eluted. Electrophoretic analysis resulted in the detection of Gal1(1-134) with a molecular weight of 14500 Da in adsorbed fractions forboth the culture supernatant and the cell extract. The adsorbedfractions from the culture supernatant and the cell extract weredialyzed separately and thoroughly against an assay medium, and thensubjected to DRG assay. Thus, a high nerve regeneration-promotingactivity was detected at a concentration of 5-50 μg/ml that wasestimated by the degree of gel staining. These results showed thepresence of Gal1 (1-134) with high nerve regeneration-promotingactivity, which was expressed in COS1 cells, in the culture supernatantand in the cells.

Example 11

Construction of E. coli Expression Vector for Gal1 (1-134)

Gal1 (1-134) was expressed using an E. coli expression vector, pET-3d(STRATAGENE), as described below. First, using pEFGal1 as a template andusing two PCR primers, HLEG12, and HLEG14, PCR was performed for 25cycles after 5 minutes at 94° C., each cycle consisting of 30 seconds at94° C./30 seconds at 60° C./1 minute at 72° C. for synthesis; followedby 5 minutes at 72° C. for reaction. An amplified fragment containing asequence spanning from the initiation codon to the termination codon ofhuman galectin-1 was digested with NcoI and BamHI, and subjected toelectrophoresis using 0.8% agarose gel, thereby yielding a fragment ofapproximately 420 bp. This fragment was purified using Prep-A-Gene DNAPurification Kit, and then linked to pET-3d that had been digested withNcoI and BamHI. E. coli DH5α was used as a host cell. A clone pETGal1(1-134) having the correct nucleotide sequence of human galectin-1 cDNA(the nucleotide sequence ranging from NcoI to BamHI of the vector isshown in SEQ ID NO:7) was selected by analysis of the nucleotidesequence. The plasmid DNA of the clone was extracted using GFX™ MicroPlasmid Prep Kit (manufactured by Pharmacia Corporation), and thentransformed into Epicurian Coli BL21 (DE3) Competent Cells (manufacturedby STRATAGENE; having T7 polymerase downstream of lac UV5 of LysogenicLambda phage). The resultant E. coli transformant was used forexpressing Gal1 (1-134). The PCR primers used herein are as follows:

-   HLEG12: 5′-AGAGTGGATCCTTATCAGTCAAAGGCCACACATTTG-3′ (SEQ ID NO: 31;    BamHI site was added at the 5′ end of the complementary strand to    GENBANK ACCESSION NO. J04456, 436-457);-   HLEG14: 5′-GAGAGACCATGGCTTGTGGTCTGGTCGC-3′ (SEQ ID NO:32; NcoI site    was added at the 5′ end of the complementary strand to GENBANK    ACCESSION NO. J04456, 50-69).

Example 12

Purification of Gal1 (1-134) Expressed in E. coli and Confirmation ofits Activity

Gal1 (1-134) was obtained from the E. coli transformant, into which theE. coli expression plasmid pETGal1 (1-134) having human galectin-1 cDNAhad been introduced. Then the presence or absence of nerveregeneration-promoting activity in Gal1 (1-134) was confirmed.

The transformant obtained in Example 11 was streaked onto the surface ofan LB agar plate containing 50 μg/ml of ampicillin, cultured overnightat 37° C., allowing the colony formation. One of these colonies wasshake-cultured in 50 ml of LB medium containing 50 μg/ml of ampicillin.The culture was added into 1000 ml of LB medium containing ampicillin at50 μg/ml allowing the initial OD₆₀₀ to be 0.2, and then shake-culturedat 37° C. until the OD₆₀₀ reached 0.5-0.6. Subsequently, IPTG was addedto the medium to a final concentration of 0.1 mM, followed byshake-culture for 3 hours, thereby inducing the expression of Gal1(1-134).

One L of the cultured cell solution was centrifuged at 10000 G for 30minutes, thereby obtaining a Gal1 (1-134) expressing bacterial pellet.The pellet was suspended in 20 ml of PBS, and then ultrasonicated withcooling. The suspension with disrupted cells was centrifuged at 10000 Gfor 30 minutes, and then the supernatant containing Gal1 (1-134) wascollected as a soluble protein. The collected supernatant was dialyzedovernight against 2 L of 20 mM Tris-HCl buffer (pH 8.0). Then thedialysate was loaded at a flow rate of 2 ml/min at room temperature to aShodex IEC DEAE-2025 column (manufactured by Showa Denko K.K., Japan; φ2 cm×15 cm) equilibrated with 0% B using 20 mM Tris-HCl buffer (pH 8.0)as a solvent A and the same buffer containing 500 mM NaCl as a solventB. After loading, the column was eluted in a 60 minutes linear gradientfrom 0% B to 30% B. The eluate was fractionated into 4 ml each, and theneach fraction was subjected to electrophoretic analysis. A band that wassuspected to be Gal1 (1-134) of a molecular weight of 14500 Da, wasdetected in a fraction with approximately 80 mM NaCl concentration. Thisfraction (15 ml) was used for the next step. Since the amino acidsequence of Gal1 (1-134) contains 6 cysteines, there is a highpossibility that SS bonds were formed in human galectin-1 which wascontained in the culture supernatant of pRLF-transfected COS1 cells andhad a nerve regeneration-promoting activity. Hence, refolding of SSbonds was performed using copper sulfate as an oxidant. Gal1 (1-134)fraction (15 ml) from the DEAE column was diluted 20-fold with 20 mMTris-HCl buffer (pH 8.0), and 1% copper sulfate solution was added tothe diluted solution at the final concentration of 0.0001%. Then thesolution was allowed to stand overnight at 4° C. 300 ml of the reactionsolution was concentrated using a ultrafiltration unit (manufactured byAmicon; membrane YM 3 with 76 mm diameter), followed by buffer exchangeusing 20 mM sodium acetate (pH 5.0). Then the solution (final volume 50ml) was loaded at a flow rate of 0.5 ml/min and at room temperature to aTSKgel SP-5PW column (manufactured by TOSOH CORPORATION, Japan; φ7.5mm×75 mm) equilibrated with 0% B, using 20 mM sodium acetate buffer (pH5.0) as a solvent A and the same buffer containing 500 mM NaCl as asolvent B. After loading, the column was eluted with a 60 minutes lineargradient from 0% B to 40% B. The eluate was fractionated into 1 ml each,and each fraction was subjected to electrophoretic analysis. A band thatwas suspected to be Gal1 (1-134) of a molecular weight of 14500 Da, wasdetected in a fraction with approximately 150 mM NaCl concentration.This fraction was used for the next step. Gal1 (1-134) fraction (8 ml)from the SP column was loaded at a flow rate of 2.0 ml/min and at roomtemperature to a YMC PROTEIN RP column (manufactured by YMC; φ 10 mm×250mm) equilibrated with 40% B, using 0.1% TFA as a solvent A and 80%acetonitrile containing 0.1% TFA as a solvent B. After loading, thecolumn was eluted with a 60 minutes linear gradient from 40% B to 50% B.The eluate was fractionated into 2 ml each, and each fraction wassubjected to electrophoretic analysis. Two bands that were suspected tobe Gal1 (1-134) having a molecular weight of 14500 Da, were detectedrespectively in two fractions, one with approximately 34% acetonitrileconcentration and the other with approximately 36%. The two bandsslightly differ in their mobility in electrophoresis with no reducingagent. However, their mobility was identical to each other inelectrophoresis after reduction treatment Accordingly, it was inferredthat the two bands were Gal1 (1-134) with different SS-bond bridges.

To confirm these bands were both Gal1 (1-134), N-terminal amino acidsequence analysis and amino acid composition analysis were performedusing the fraction eluted at approximately 36% acetonitrile. First,N-terminal amino acid sequence analysis was performed using a proteinsequencer (manufactured by Perkin-Elmer Corporation; type 492). As aresult, the following N-terminal amino acid sequence was detected(wherein X is an undetermined amino acid):

-   AlaXGlyLeuValAlaSerAsnLeuAsnLeuLysProGlyGluXLeuArgValArg (SEQ ID    NO:33)    Since this sequence was identical to the N-terminal amino acid    sequence of Gal1 (1-134), it was confirmed that the protein purified    by PROTEIN RP column was Gal1 (1-134). Next, amino acid composition    was analyzed after hydrolysis of acid using 6 N hydrochloric acid. A    sample was subjected to acid hydrolysis in vapor phase with 6 N    hydrochloric acid using a PICO•TAG workstation (manufactured by    Waters Corporation) for 1 hour at 150° C. Then the sample was    subjected to fluorescent derivatization reaction using an AccQ Fluor    reagent (manufactured by Waters Corporation). The amino acids were    analyzed using an AccQ Tag amino acid analysis system (manufactured    by Waters Corporation), and the following amino acid composition    ratio was detected. Theoretical values for Gal1 (1-134) are shown in    parenthesis:-   Asp:22.18 (22), Ser:5.51 (5), Glu:10.30 (10), Gly:11.38 (11),    His:2.12 (2), Arg:5.10 (5), Thr:3.89 (4), Ala:13.90 (14), Pro:7.09    (7), Tyr:2.08 (2), Val:9.23 (10), Met:1.15 (1), Lys:7.97 (8),    Ile:3.29 (4), Leu:11.85 (12), Phe:9.96 (10) (Cys, and Trp were not    determined).

Therefore, it was confirmed that the amino acid composition ratio of thepurified protein was in good agreement with the theoretical values ofGal1 (1-134). It was confirmed that the purified protein was Gal1(1-134), and the purity was significantly high.

Similarly, N-terminal amino acid sequence analysis and the amino acidcomposition analysis were conducted for the fraction eluted atapproximately 34% acetonitrile. As a result, it was confirmed that theprotein eluted in this fraction was Gal1 (1-134) in each analysis.Subsequently, the concentration of Gal1 (1-134) in each sample wasdetermined from the results of the amino acid analysis. The fractionswere adjusted to 0.5, 5, 50, 500, 5000 μg/ml, respectively, with anassay medium, and then the nerve regeneration-promoting activity wasmeasured by DRG assay. As a result, Gal 1(1-134) showed a nerveregeneration-promoting activity at the concentration from 0.5 μg/ml to500 μg/ml, depending on the concentration (FIG. 4). Hence, it wasconfirmed that E. coli-expressed Gal1 (1-134) in which intramolecular SSbonds were formed via oxidation process with copper sulfate possessed anerve regeneration-promoting activity.

Example 13

Identification of the Pattern of Disulfide Linkages Form of E. coliExpressed Gal1 (1-134)

The pattern of disulfide linkages of E. coli Gal1 (1-134) obtained inExample 12 (the fraction eluted at an approximately 36% acetonitrile)was identified by mass spectrometry of peptide fragments after enzymaticdigestion. 50 μl of the E. coli expressed Gal1 (1-134) fraction(equivalent to 15 μg as determined by amino acid analysis) was subjectedto centrifugal evaporation, dissolved in 20 μl of 100 mM Tris-HCl buffer(pH 6.8), and then digested with 0.6 μg of trypsin for 16 hours at 37°C.

The peptide fragments present in the digestion buffer were loaded to aSymmetry C18 reverse phase column (manufactured by Waters Corporation; φ2.0 mm×50 mm) at a flow rate of 0.25 ml/min at a column temperature of40° C. using 0.05% TFA as a solvent A and a mixture ofisoparopanol:acetonitrile=7:3 with 0.02% TFA as a solvent B. The columnwas eluted with a 50 minutes linear gradient from 1% B to 50% B, tocollect the fragments by fractionation (FIG. 5). Part (0.5 μl) of eachresultant peptide fragment solution was spotted on a sample plate, andmixed with 0.5 μl of α-cyano-4-hydroxycinnamic acid (CHCA) that had beendissolved at 10 mg/ml in a solution of acetonitrile:0.1% TFA=50:50,followed by air-drying. The dried product was subjected to massspectrometry using a mass spectrometer, Matrix-Assisted Laser DesorptionIonization ime-of-Flight (MALDI-TOF) type (manufactured by Perceptive;Voyager Elite). Fragment Nos, detected mass, and amino acid sequencesassigned based on the mass are as shown below:

TP1 786.429 G²¹EVAPDAK²⁸ (SEQ ID NO:34); TP2 534.028 F¹⁰⁸PNR¹¹¹ (SEQ IDNO:35); TP3 1042.15 V¹⁹RGEVAPDAK²⁸ (SEQ ID NO:36); TP4 1077.29D⁶⁴GGAWGTEQR⁷³ (SEQ ID NO:37); TP5 969.466 L¹⁰⁰PDGYEFK¹⁰⁷ (SEQ IDNO:38); TP6 3021.11 D³⁷SNNLC⁴²LHFNPR⁴⁸ (SEQ ID NO: 39) +F⁴⁹NAHGDANTIVC⁶⁰NSK⁶³ (SEQ ID NO:40); TP7 878.451 S²⁹FVLNLGK³⁶ (SEQ IDNO:41); TP8 1785.14 L¹¹²NLEAINYMAADGDFK¹²⁷ (SEQ ID NO:42) TP9 5222.08A¹C²GLVASNLNLKPGEC¹⁶LR¹⁸ (SEQ ID NO:43) +E⁷⁴AVFPFQPGSVAEVC⁸⁸ITFDQANLTVK⁹⁹ (SEQ ID NO:44); + C¹³⁰VAFD¹³⁴ (SEQ IDNO:45).

These results revealed that E. coli expressed Gal1 (1-134) (the fractioneluted at approximately 36% acetonitrile) had three sets of SS-bondbridges. One of them was Cys42-Cys60, and Cys88 and Cys130 were linkedwith either Cys2 or Cys 16. To identify the pattern of disulfidelinkages of the remaining two sets, secondary digestion was performed byadding lysil-endopeptidase (manufactured by WAKO PURE CHEMICALINDUSTRIES., LTD., Japan) to TP9. TP9 was subjected to centrifugalevaporation, to which 20 pmol of lysil-endopeptidase (manufactured byWAKO PURE CHEMICAL INDUSTRIES., LTD., Japan) was added, and thendigested for 16 hours at 37° C. in 20 μl of 100 mM Tris-HCl buffer (pH6.8). The peptide fragments present in the digestion buffer were loadedto a Symmetry C18 reverse phase column (manufactured by WatersCorporation; φ 2.0 mm×50 mm) at a flow rate of 0.25 ml/min at a columntemperature of 40° C., using 0.05% TFA as a solvent A and a mixture ofisoparopanol:acetonitrile=7:3 with 0.02% TFA as a solvent B. The columnwas eluted with a 30 minutes linear gradient from 1% B to 50% B tocollect the fragments by fractionation (FIG. 6). After centrifugalevaporation, the resultant peptide fragments were subjected to massspectrometry using a mass spectrometer, type MALDI-TOF (manufactured byPerceptive; Voyager Elite). Fragment Nos, detected mass, and amino acidsequences assigned based on the mass are as shown below:

TPAP1 1755.31 A¹C²GLVASNLNLK¹² (SEQ ID NO:46) + C¹³⁰VAFD¹³⁴ (SEQ ID NO:47); TPAP2 3484.21 P¹³GEC¹⁶LR¹⁸ (SEQ ID NO:48) +E⁷⁴AVFPFQPGSVAEVC⁸⁸ITFDQANLTVK⁹⁹ (SEQ ID NO:49).

It was thus identified that the pattern of disulfide linkages of theremaining two sets were Cys2-Cys130 and Cys16-Cys 88. As a result, itwas found that E. coli expressed Gal1 (1-134) (the fraction eluted atapproximately 36% acetonitrile) had three sets of SS bonds and thepattern of disulfide linkages was Cys42-Cys60, Cys2-Cys130, andCys16-Cys 88.

Similarly, the pattern of disulfide linkages was identified for E. coliexpressed Gal1 (1-134) that had been eluted at approximately 34%acetonitrile. The SS-linkage form was a mixture of the form: Cys2-Cys42,Cys16-Cys88, and Cys60-Cys130 and the form: Cys2-Cys60, Cys16-Cys88 andCys42-Cys130.

Example 14

Measurement of Lectin Activity of E. coli Expressed Gal1 (1-134)

Galectin belongs to a protein family, generally called animal lectin,and binds to sugars containing P-galactoside. To determin if the E. coliexpressed Gal1 (1-134) possesses a lectin activity (a β-galactosidebinding activity), two methods, i.e. an affinity chromatography usingβ-galactoside as a ligand and a hemagglutination assay, were conducted.

(1) Affinity Chromatography Using β-Galactosid as a Ligand

The E. coli expressed Gal1 (1-134) was loaded at a flow rate of 0.5ml/min and at 4° C. to a lactose agarose column (manufactured by HONENCORPORATION; φ 5.0 mm×50 mm) that had been equilibrated with 0% B, usingPBS as a solvent A and PBS containing 0.1 M lactose as a solvent B.After loading, the column was eluted with a 30 minutes linear gradientfrom 0% B to 100% B. The eluate was fractionated into 2 ml each, andseparately analyzed by electrophoresis. As a result, Gal1 (1-134) with amolecular weight of 14500 Da was detected in flow-through fractions. Inaddition the same sample was subjected to a reduction treatment with 5mM DTT for 2 hours at room temperature, and applied to the same lactoseagarose column. In this time, Gal1 (1-134) was not detected inflow-through fractions, but in fractions having about 10 mM lactose.These results confirmed that the E. coli expressed Gal1 (1-134) couldnot bind to β-galactoside (lactose), but the β-galactoside bindingactivity is restored by reduction treatment.

(2) Measurement of Hemagglutination Activity

5 ml of PBS was added to 1 ml of stored rabbit blood (manufactured byCosmo Bio), followed by centrifugation at 2000 G for 5 minutes, therebyremoving the supernatant. This procedure was repeated 3 times to obtainan erythrocyte fraction. Then 4.9 ml of PBS was added to 100 μl of theerythrocyte fractions so as to prepare 2% blood cell suspension. 50 μlof E. coli expressed human galectin-1 (diluted 50 μg/ml with PBS)obtained in Example 12 was added to the first row of a 96-well titerplate; and 50 μl each of the same galectin-1 2-fold diluted (at maximum0.39 μg/ml), was added to the second and the following rows. Then 50 μleach of 2% blood cell suspension was dispensed into each well, andallowed to stand for 1 hour at room temperature. At this time, the samedilution series were prepared for Concanavalin A and for E. coliexpressed Gal1 (1-134) that had been subjected to a reduction treatmentwith concanavalin A and 5 mM DTT for 2 hours at room temperature, andsimultaneously the hemagglutination activity was measured. As a result,hemagglutination activity was detected at a concentration of 6.25 μg/mlor more for Concanavalin A and for any of E. coli expressed Gal1 (1-134)that had been subjected to reduction treatment with 5 mM DTT for 2 hoursat room temperature. However, for E. coli expressed Gal1 (1-134) (notreduced) obtained in Example 12, no hemagglutination activity wasdetected even at a concentration of 50 μg/ml (FIG. 7).

As a result of both the affinity chromatography using β-galactoside as aligand and the measurement of hemagglutination activity, it wasconfirmed that Gal1 (1-134) having SS bonds, as prepared in Example 12,possessed no lectin activity.

Example 15

Preparation and Purification of Anti-Gal1 (1-134) Antiserum

A rabbit was immunized with E. coli expression Gal1 (1-134) as anantigen to obtain a rabbit anti-Gal1 (1-134) antiserum. The E. coliexpression Gal1 (1-134) as the antigen was purified by a TSKgel SP-5PWcolumn (manufactured by TOSOH CORPORATION) as described in Example 12.Two rabbits were immunized. After emulsion was prepared using Gal1(1-134) and oil adjuvant, it was administered subcutaneously to a rabbitin a dose of 20-200 μg, 6 times in total over approximately 2 months.After immunization, exsanguination was performed, thereby obtaining aserum of approximately 75 ml per rabbit. Then titration was conducted byELISA using plates with antigens immobilized thereon. The obtainedantiserum showed a significant value even if it was diluted 1: 204800 atthe maximum. That is, a highly specific and good antiserum was obtained.

The resulting antiserum was purified using a protein A column, andimmunoglobulin (IgG) fractions were prepared. 10 ml of the antiserum wasdiluted 2-fold with 10 ml of PBS, filtered, then applied to a HiTrapProtein A column (manufactured by Pharmacia; 1 ml gel) equilibrated withPBS. After washing with 20 ml of PBS, adsorbed fractions were elutedusing 5 ml of 100 mM glycine-HCl buffer (pH 2.7). At this time, to thefraction tubes, 500 μl of 1 M Tris-HCl buffer (pH 9.0) was previouslyadded. Plow-through fractions, as well as adsorbed fractions, wereanalyzed by electrophoresis. Only IgG was detected in the adsorbedfractions. Since the flow-through fractions also contained IgG, theywere purified again in the same way as described above. When thepurified product was analyzed by electrophoresis, second flow-throughfractions also contained IgG. The second fractions were similarlyre-purified. The adsorbed fractions from three purification runs werecombined (16.5 ml), and were dialyzed against 2 L of PBS overnight,thereby obtaining approximately 30 mg of anti-Gal1 (1-134) IgG (as aprotein concentration determined by Coomassie dye binding method).

Example 16

Preparation of Anti-Gal1 (1-134) IgG Column

Anti-Gal1 (1-134) IgG column was prepared by immobilizing anti-Gal1(1-134) IgG obtained in Example 15 to a resin. 5 ml of a solution of 2mg/ml anti-Gal1 (1-134) IgG was concentrated using a ultrafiltrationmembrane (10,000 cut-off, MILLIPORE), and simultaneously buffer exchangewas performed with 1 ml of a coupling buffer (0.2 M sodium bicarbonate,0.5 M sodium chloride, pH 8.3). A HiTrap NHS-activated column(manufactured by Pharmacia; 1 ml gel) was washed with 6 ml of 1 mMhydrochloric acid, and then to which 1 ml of the above IgG solution wasadded, allowing to stand for 30 minutes at room temperature.Subsequently, the column was washed with 3 ml of the same couplingbuffer, and washed with 6 ml each of a washing buffer A (0.5 M ethanolamine, 0.5 M sodium chloride, pH 8.3) and a washing buffer B (0.1 Msodium acetate, 0.5 M sodium chloride, pH 4.0). Further, 6 ml of thewashing buffer A was loaded to the column, and then allowed to stand for30 minutes at room temperature. The column was washed with 6 ml each ofwashing buffer B, washing buffer A, washing buffer B, and PBS, in order,so that anti-Gal1 (1-134) IgG column was prepared.

Example 17

Purification of COS1 Cell Expressed Gal1 (1-134) and Identification ofthe Pattern of Disulfide Linkages

To determine the pattern of SS linkages of Gal1 (1-134) expressed inCOS1 cells and secreted in culture supernatant, Gal1 (1-134) waspurified from the culture supernatant of COS 1 cells by a purificationtechnique not employing a lactose agarose column after reductiontreatment as described in Example 10. Plasmid pEFGal1 having humangalectin-1 cDNA as described in Example 9 was transfected in the samemanner as described in Example 7, and 250 ml of the culture supernatantwas obtained. The culture supernatant was added at a flow rate of 0.5ml/ml and at 4° C. to the anti-Gal1 (1-134) IgG column obtained inExample 16 that had been equilibrated with PBS, using a Peristaltic pump(manufactured by Pharmacia; type P-1). The column was washed with 50 mlof PBS, and then adsorbed fractions were eluted with 3 ml of 100 mMglycine-HCl buffer (pH 2.7). To the fraction tubes, 300 μl of 1MTris-HCl buffer (pH 9.0) was previously added. When the adsorbedfraction was analyzed by electrophoresis, a band suspected to be Gal1(1-134) with a molecular weight of 14500 Da was detected. 3.3 ml of thisfraction was used for the next step. Using 0.1% TFA as a solvent A, and80% acetonitrile containing 0.1% TFA as a solvent B, the anti-Gal1(1-134) IgG column adsorbed fraction was loaded at a flow rate of 0.5ml/min at room temperature to a YMC PROTEIN RP column (manufactured byYMC; φ 4.6 mm×150 mm) equilibrated with 40% B. After loading, the columnwas eluted with a 45 minutes linear gradient from 40% B to 55% B. Theeluate was fractionated into 0.5 ml each, and then each fraction wasanalyzed by electrophoresis. Thus only the band suspected to be Gal1(1-134) with a molecular weight of 14500 Da was detected in the fraction(Fr35) having approximately 38% acetonitrile. 0.5 μl of this fractionwas subjected to mass spectrometry using a MALDI-TOF mass spectrometer(manufactured by Perceptive; voyager Elite) in the same manner asdescribed in Example 13.

As a result, the molecular mass of this band was 14622.1 Da. Compared tothe molecular mass of 14583.4 Da of Gal1 (1-134) being in the reducedstate, it was found that this band corresponded to Gal1 (1-134) whereinthe N-terminus was acetylated and SS bonds were formed. Thus, thepurification of Gal1 (1-134) expressed in COS1 cells and secreted in theculture supernatant was completed.

Because it was predicted from the results of mass spectrometry that Gal1(1-134) secreted in the culture supernatant carries is linked by SSbonds, the patterns of disulfide linkages were identified. Briefly, theGal1 (1-134) was digested with trypsin and lysyl-endopeptidase accordingto the methods described in Example 13. The resulting fragments weresubjected to mass spectrometry, and each fragment was assigned. It wasfound that the pattern of SS linkages form of Gal1 (1-134) secreted inthe culture supernatant was Cys42-Cys60, Cys2-Cys130, and Cys16-Cys88.This pattern was identical to that of E. coli expressed Gal1 (1-134)(the fraction eluted at approximately 36% acetonitrile) as described inExample 13.

Example 18

Sciatic Nerve Injury Model Experiment

Under chloral hydrate anesthesia, sciatic nerves of an adult BALB/cmouse (female, 3-6 weeks old) were exposed at the femoral region andtransected. The transected nerve site, at 7 mm from the transectedcentral side end, was injured with forceps. An osmotic minipump (Alzet,model 2002 for 2 weeks, or model 2001 for 1 week) was placed at asubcutaneous position of the murine back, and then the transectedcentral side end of the sciatic nerve was inserted into a polyethylenetube connected to a pump. The mini pump was previously filled with 220μl a solution of 5 μg/ml Gal1 (1-134) in physiological saline. This Gal1solution was continuously sent into the transected nerve end at 1.0 or0.5 μl/h over 14 days or 7 days. For the 14 days model, the region offrom the injured site to the transected nerve end was further injured byfreezing. On day 7 and on day 14 after injury, the injured sciatic nervewas fixed by perfusion with 4% paraformaldehyde, removed, and thensubjected to 2.5% glutaraldehyde fixation. After osmium fixation, thenerve was epon-embedded, sectioned into an ultra-thin section, and thenobserved it with an electron microscope. A site, at 6 mm away from theinjured site and 1 mm from the transected central side end (i.e., thesite administered with the solution), was observed with an electronmicroscope.

On day 7, for Gal1 (1-134) administered groups, there were observedtendencies of the increase in myelin phagocytes and of the increase inregenerating axons that was thought of an initial regeneration processof medullary sheath, compared to control, suggesting early disruption ofmyelin and progression of nerve regeneration. On day 14, for controlgroups, many degenerative myelin remained and regenerating myelinatednerves were hardly observed (FIG. 8B). On the other hand, for Gal1(1-134) administered groups, a decrease was observed both in myelinphagocytes and in degenerative myelin, and regeneration of lots ofthinly myelinated nerves between regenerated non-medullary nerve bundleswas observed (FIG. 8A). These findings suggest that Gal1 (1-134)administration results in promoted regeneration of injured nerves.

Example 19

Preparation of Collagen Gel Silicon Chamber for Regeneration Experimentof Transected Peripheral Nerve and Regeneration Experiment of TransectedPeripheral Nerve

One end of a sterilized silicon tube (outer diameter 1.5 mm, innerdiameter 1 mm, and length 7 mm) was closed with a sterilized glass bead.0.8 ml of collagen solution (0.3%) was extracted from a rat tail at 0°C. and dissolved in 0.1% acetic acid, followed by sequential addition of10 μl of Gal1 (1-134) at 500 ng/ml, 0.1 ml of 10×NEM (GIBCO, MinimumEssential Medium), and 0.1 ml of a pH adjusting solution (0.477 g Hepesdissolved in 10 ml 0.3 N NaOH). Similarly, a Gal1 (1-134)-free controlcollagen solution was prepared. Silicon tubes were filled on ice witheach collagen solution, and collagen solutions were converted to a gelphase by warming at 37° C., thereby preparing collagen gel siliconchambers.

A 10-11 weeks old male Wister rat was anesthetized with pentobarbital(0.3 ml, 50 mg/ml). Then the femoral region was incised to expose thesciatic nerve and the nerve was separated into fibular nerve and tibialnerve. Collagen gel silicon chambers containing Gal1 (1-134) and controlcollagen gel silicon chambers were prepared. The Gal1 (1-134)-containingchamber was arranged in parallel to the left fibular nerve, with theiropening in proximity to the left fibular nerve, and the both ends werefixed to the muscle with stitches. Next the fibular nerve wastransected, the transected proximal nerve ending was put in the silicontube, and then the nerve fibers were fixed to the tube with stitches.The incised site was sutured. Similarly, the right fibular nerve wastransected, and the control chamber was fixed thereto. Gal1(1-134)-containing chamber and control chamber were fixed, whilealternating the sides (left and right) per rat. 7 and 10 days afterrearing, the rat was anesthetized with Pentobarbital, and then fixed byperfusion with a zamboni fixation solution. Then the transplantedchamber was removed. Tubes were fixed with 4% paraformaldehyde, and thenlongitudinal or cross-frozen sections were generated in a cryostat.Next, immunostaining was performed using hematoxylin-eosin (HE) stainingand anti-neurofilament (NF) antibody, and anti-S 100 antibody. Based onimages of immuno-staining of longitudinal sections with HE and withanti-S100 antibody and anti-NF antibody, a migration distance from thetransected nerve end in the silicon tube to S100 positive Schwann cells,and an elongation distance of NF positive regenerated-axons, weremeasured. The number of NF positive regenerated-axons was counted fromcross sections.

Based on the results of HE staining, the migration distance of cellsmigrated from the transected nerve end into the gel was measured. Asshown in FIG. 9, the presence of Gal1 (1-134) resulted in the increasein migration distance of migrating cells when compared to the control.Specifically, as shown in Table 1, on day 7 after treatment, the averagemigration distance of the control was 0.7 mm while that of the Gal1(1-134)-administered group was 1.1 mm; on day 10, that of the controlwas 1.2 mm while that of Gal1 (1-134)-administered group was 1.9 mm.That is, administration of Gal1 (1-134) promoted migration of cells.S100 positive Schwann cell, a major migrating cell, reached the tip ofthe regeneration site. Further, as shown in FIG. 10, NF positiveregenerating axons extended to this position of Schwann cell. It wasalso found that the number of axons was increased by Gal1 (1-134). Table2 shows the measurements of the number of regenerating axons usingcross-sectioned sections at two positions, 0.5 mm and 1.0 mm from thetransected nerve end. Average number of regenerating axons at theposition 0.5 mm from the transected nerve end was 241 for the control,and 882 for the Gal1 (1-134)-administered group; that at 1.0 mm from thetransected nerve end was 52 for the control, but as high as 302,significantly increased for the Gal1 (1-134)-administered group. Asdescribed above, in the Gal1 (1-134)-administered group, migration ofcells, mainly Schwann cell, from the transected nerve end was promoted,as well as the elongation of regenerating axons was promoted and thenumber of the same was increased. It was clearly shown that Gal1 (1-134)was a useful in vivo as a nerve regeneration-promoting factor.

TABLE 1 Migration distance of migrating cells from the transected nerveend Average migration distance of aletocyte from transected end DayNumber of tests (mm) Control Day 7 11 0.7 ± 0.2 Day 10 8 1.2 ± 0.4 Gal1(1-134) day 7 5 1.1 ± 0.1 Day 10 7 1.9 ± 0.5 Unpaired Student's t-test:day 7: p < 0.001; day 10:p < 0.01.

TABLE 2 Number of neurofilament positive axons Distance from transectednerve end Test number 0.5 mm 1.0 mm Control No. 134 126 50 No. 135 17344 No. 158 489 167 No. 159 157 41 No. 161 242 68 No. 164 395 0 No. 182108 0 control average 241 ± 135 52 ± 52 Gal1 (1-134) No. 139 894 555 No.160 444 126 No. 163 387 86 No. 171 1374 795 No. 174 432 137 No. 175 1766114 Gal1 (1-134) average 882 ± 526 302 ± 273 Unpaired Student's t-test:0.5 mm:p < 0.01; 1.0 mm:p < 0.05.

Example 20

Construction of E. coli Expression Vector for a Fusion Protein ofGlutathione-S-Transferase (GST) and Human Galectin-1 (Amino Acids 1-134)(Hereinafter, Referred to as “GST-Gal1 (1-134)”

GST-Gal1 (1-134) was expressed as described below using pGEX-5X-2(manufactured by Pharmacia), an expression vector for a fusion proteinwith glutathione-S-transferase (GST). First, PCR was performed usingpEFGal1 as a template and using two PCR primers, HLEG11 and HLEG13. PCRwas performed for 25 cycles after 5 minutes at 94° C., each cycleconsisting of 30 seconds at 94° C./30 seconds at 60° C./1 minute at 72°C. for synthesis; followed by 5 minutes at 72° C. for reaction. Theamplified fragment was digested with BamHI and NotI, subjected toelectrophoresis using 0.8% agarose gel, thereby collecting a fragment ofapproximately 420 bp. The fragment was purified using Prep-A-Gene DNAPurification Kit, and linked to pGEX-5X-2 that had been digested withBamHI and NotI. E. coli DH5 α was used as a host cell. A clone pGEXGal1(1-134) having a correct nucleotide sequence of human galectin-1 cDNA(SEQ ID NO:8; the nucleotide sequence from BamHI to NotI of the vector)was selected by analysis of the nucleotide sequence. The selected clonewas used as a transformant for expressing GST-Gal1 (1-134). Sequences ofthe PCR primers used are as follows:

-   HLEG11: 5′-GAGAGAGGATCCCCATGGCTTGTGGTCTGGTCGC-3′ (SEQ ID NO: 50;    BamHI site was added to the 5′ end of GENBANK ACCESSION NO. J04456,    50-69, so that the primer could be linked to GST-Tag in frame);-   HLEG13: 5′-AGAGTGCGGCCGCTTATCAGTCAAAGGCCACACATTTG-3′ (SEQ ID NO: 51;    NotI site was added to the 5′ end of GENBANK ACCESSION NO. J04456,    436457).

This expression plasmid contained GST protein followed by factor Xarecognition sequence, and a sequence encoding human galectin-1 (aminoacids 1-134) [The amino acid sequence from the factor Xa recognitionsequence to human galectin-1 (amino acid 1-134) is shown in SEQ IDNO:9.].

Example 21

Construction of E. coli Expression Vector for a Fusion Protein ofGlutathione-S-Transferase (GST) and Human Galectin-1 (Amino Acid 1-134)in Which Cys at Position 2 was Converted to Ser

GST-Gal1 (2/Ser) was expressed as described below using pGEX-5X-2(manufactured by Pharmacia), an expression vector for a fusion proteinwith glutathione-S-transferase (GST). First, PCR was performed usingpGEXGal1 (1-134) as a template and using two PCR primers, HLEG15 andHLEG23. PCR was performed for 25 cycles after 5 minutes at 94° C., eachcycle consisting of 30 seconds at 94° C./30 seconds at 55° C./1 minuteat 72° C. for synthesis; followed by further 5 minutes at 72° C. Theamplified fragment was digested with BamHI and NotI, subjected toelectrophoresis using 2% agarose gel, thereby collecting a fragment ofapproximately 420 bp. The fragment was purified using Prep-A-Gene DNAPurification Kit, and linked to pGEX-5X-2 that had been digested withBamHI and NotI. E. coli DH5 was used as a host cell. A clone pGEXGal1(2/Ser) having a nucleotide sequence of human galectin-1 cDNA, in whichnucleotide No. 56 (GENBANK ACCESSION NO. J04456) had been converted toA, and nucleotide No. 58 (GENBANK ACCESSION NO. J04456) to C (thenucleotide sequence from BamHI to NotI of the vector was the same asthat in SEQ ID NO:8, except that T at the 15-position was changed to A,T at the 17-posiiton to C, respectively) was selected by analysis of thenucleotide sequence. The selected clone was used as a transformant forexpressing GST-Gal1 (2/Ser). Sequences of the PCR primers used hereinare as follows:

-   HLEG15: 5′-GAGAGAGGATCCCCATGGCTAGCGGTCTGGTCG-3′ (SEQ ID NO: 64)    (BamHI site was added to the 5′ end of GENBANK ACCESSION NO. J04456,    50-68, so that the primer could be linked in frame to GST-Tag. In    addition, nucleotide No. 56 was converted to A, and 58 to C).-   HLEG23: 5′-AGAGAGCGGCCGCTTATCAGTCAAAGGCCACACATTT-3′ (SEQ ID NO: 53;    NotI site was added to the 5′ end of the complementary strand of    GENBANK ACCESSION NO. J04456, 437-457).

This expression plasmid contained GST protein followed by factor Xarecognition sequence, and a sequence encoding human galectin-1 variant[The amino acid sequence from the factor Xa recognition sequence to thehuman galectin-1 variant was the same as shown in SEQ ID NO:9 exceptthat Cys at the 10-position was changed to Ser].

Example 22

Construction of E. coli Expression Vector for a Fusion Protein(Hereinafter Referred to as “GST-Gal1 (all/Ser)”) ofGlutathione-S-Transferase (GST) and Human Galectin-1 (Amino Acids 1-134)in Which all Cys Residues Were Converted to Ser Residues

GST-Gal1 (all/Ser) was expressed as described below using pGEX-5X-2(manufactured by Pharmacia), an expression vector for a fusion proteinwith glutathione-S-transferase (GST). Six Cysteine residues were presentin human galectin. First, to convert Cys at the 130-position in SEQ IDNO:1 to Ser, two synthetic oligomers, HLEG21 and HLEG22, were annealed(5 minutes at 99° C./5 minutes at 80° C./5 minutes at 70° C./5 minutesat 60° C./5 minutes at 50° C./5 minutes at 40° C./5 minutes at 30° C.),and then linked to pGEX-5X-2 that had been digested with EcoRI and NotI.E. coli DH5 was used as a host cell. Of nucleotide sequences downstreamof EcoRI located at nucleotide No. 366 of human galectin-1 cDNA (GENBANKACCESSION NO. J04456), clone pGEXGal1 (all/Ser-3′) (a sequence fromEcoRI site to Not I site is shown in SEQ ID NO:10) having a sequencewherein T of nucleotide No. 440 (GENBANK ACCESSION NO. J04456) wasconverted to A, T of nucleotide No. 442 (GENBANK ACCESSION NO. J04456)to C, was selected by analysis of the nucleotide sequence. Syntheticoligomer sequences used are as follows:

-   HLEG21:    5′-AATTCAAGTTCCCCAACCGCCTCAACCTGGAGGCCATCAACTACATGGCAGCTGACGGTGACTTCAAGATCAAAAGCGTGGCCTTGACTGATAAGC-3′    (SEQ ID NO: 54; Not I site was added to the 3′ end of GENBANK    ACCESSION NO. J04456, 366-457; T of nucleotide No. 440 was converted    to A, T of nucleotide No. 442 to C;).-   HLEG22:    5′-GGCCGCTTATCAGTCAAAGGCCACGCTTTTGATCTTGAAGTCACCGTCAGCTGCCATGTAGTTGATGGCCTCCAGGTTGAGGCGGTTGGGGAACTTG-3′    (SEQ ID NO:55; NotI site was added to the 5′ end of the    complementary strand of GENBANK ACCESSION NO. J04456, 370-457;    Antisense, A, of nucleotide No. 440, was converted to T, and    antisense, A, of nucleotide No. 442, to G).

The remaining 5 Cysteine residues were converted to Serine residues asdescribed below. PCR was performed using 2 ng of pGEXGal1 (1-134) as atemplate and using 5 pmol each of synthesized primers, HLEG16 andHLEG18. Briefly, the PCR was performed in a volume of 50 μl by GeneAmp™PCR System 2400 (manufactured by Perkin-Elmer Corporation) usingAmpliTaq™ DNA polymerase (manufactured by Perkin-Elmer Corporation). Thereaction cycle was repeated 25 times after 5 minutes at 94° C., eachconsisting of 30 seconds at 94° C./2 minutes at 72° C. for synthesis;followed by further 5 minutes at 72° C. Furthermore, PCR was performedunder the same conditions above using pGEXGal1(1-134) as a template andusing 5 pmol each of synthesized primers, HLEG17 and HLEG20. Aftermixing the above two reaction solutions, PCR reaction was performed for5 times, each cycle consisting of 30 seconds at 94° C. and 2 minutes at72° C. for synthesis. Then PCR was performed in a volume of 100 μl using1 μl of the resulting reaction solution as a template, and using 20 pmoleach of synthesized primers, HLEG15 and HLEG19. The PCR reaction wasperformed for 25 times, each consisting of 30 seconds at 94° C./30seconds at 55° C./1 minute at 72° C. for synthesis; followed by further5 minutes at 72° C. The amplified fragment was digested with EcoRI andBamHI, subjected to electrophoresis with 2% agarose gel, therebyrecovering a fragment of approximately 330 bp. Then the fragment waspurified with Prep-A-Gene DNA Purification Kit, and then linked topGEXGal1 (all/Ser-3′) that had been digested with EcoRI and BamHI. E.coli DH5 was used as a host cell. Clone, pGEXGal1 (all/Ser), having anucleotide sequence of human galectin-1 wherein all Cys had beenconverted to Ser (i.e., a nucleotide sequence from BamHI to NotI of thevector was the same as shown in SEQ ID NO:8 except that T at the15-position was converted to A, T at the 17-position to C, T at the57-position to A, T at the 135-position to A, T at the 189-position toA, T at the 273-position to A, T at the 399-position to A, and T at the401-position to C), was selected by analysis of the nucleotide sequence.Sequences of the PCR primers used are as follows:

-   HLEG16:    5′-ATGGCTAGCGGTCTGGTCGCCAGCAACCTGAATCTCAAACCTGGAGAGAGCCTFCG-3′ (SEQ    ID NO:56; GENBANK ACCESSION NO. J04456, 50-105. Nucleotide No. 56    was converted to A, No. 58 to C, and No. 98 to A);-   HLEG17:    5′-ACCTGAGCCTGCACTTCAACCCTCGCTTCAACGCCCACGGCGACGCCAACACCATCGTGAGCAAC-3′    (SEQ ID NO:57; GENBANK ACCESSION NO. J04456, 171-235. Nucleotide    No.176 was converted to A, and No. 230 to A);-   HLEG18:    5′-GTTGCTCACGATGGTGTTGGCGTCGCCGTGGGCGTTGAAGCGAGGGTTGAAGTGCAGGCTCAGGT-3′    (SEQ ID NO:58; complementary strand of GENBANK ACCESSION NO. J04456,    171-235. Antisense, A, of nucleotide No. 176, was converted to T,    and antisense, A, of No. 230, to T);-   HLEG19: 5′-AACTTGAATTCGTATCCATCTG-3′ (SEQ ID NO:59; complementary    strand of GENBANK ACCESSION NO. J04456, 354-375);-   HLEG20:    5′-AACTTGAATTCGTATCCATCTGGCAGCTTGACGGTCAGGTTGGCCTGGTCGAAGGTGATGCTCAC-3′    (SEQ ID NO:60; complementary strand of GENBANK ACCESSION NO. J04456,    311-375. Antisense, A, of nucleotide No. 314, was converted to T).

The expression plasmid contained a sequence encoding GST proteinfollowed by a factor Xa recognition sequence, and a sequence encodinghuman galenctin-1 in which all the cysteine residues were converted toSerine residues (The amino acid sequence from the factor Xa recognitionsequence to human galectin-1 variant was the same as shown in SEQ ID NO:9 except that all the cysteine residues were converted to serineresidues).

Example 23

Expression of GST Fusion Protein (GST-Gal1 (1-134)) in E. coli andRemoval of GST Portion by Factor Xa, and Purification of Gal1 (1-134)[GIPM-Gal1 (1-134)] Having GlyIleProMet (SEQ ID NO: 61) Added to theN-Terminus

First, the clone obtained in Example 20 was streaked onto the surface ofan LB agar plate containing 50 μg/ml of ampicillin, and then culturedovernight at 37° C., allowing the colony formation. One of the resultingcolonies was shake-cultured in 50 ml of an LB medium containing 50 μg/mlof Ampicillin. Subsequently the culture was added to 1000 ml of an LBmedium containing 50 μg/ml of ampicillin at an initial OD₆₀₀ of 0.2, andshake-cultured at 37° C. until the OD₆₀₀ reached 0.5-0.6. Then IPTG(isopropylthiogalactosid) was added at a final concentration of 0.1 mM,followed by 3 hours shake-cultur, thereby inducing expression ofGST-Gal1 (1-134). One L of the cell culture solution was centrifuged at10000 G for 30 minutes to obtain a pellet of cells expressing GST-Gal1(1-134) fusion protein. The pellet was suspended in 20 ml of PBS, andsonicated with cooling so that the cells were disrupted. Thecell-disrupted solution was centrifuged at 10000 G for 30 minutes,thereby collecting the supernatant containing GST-Gal1 (1-134) fusionprotein as a soluble protein. The collected supernatant was loaded to aglutathione-Sepharose 4B column (manufactured by Pharmacia; φ 3 cm×5 cm)equilibrated with PBS. The elution buffer was exchanged with 50 mMTris-HCl buffer (pH 8.0) containing 1 mM calcium chloride and 100 mMNaCl, for the sake of thorough washing. Then 400 units factor Xa wasloaded to the column which was allowed to stand overnight at roomtemperature. Next, GIPM-Gal1 (1-134) that had been cleaved with thefactor Xa was eluted at a flow rate of 1 ml/min with 50 mM Tris-HClbuffer (pH 8.0) containing 1 mM calcium chloride and 100 mM NaCl. Theeluate was fractionated into 3 ml each and each of the fractions wasanalyzed with electrophoresis. The fraction in which a band suspected tobe GIPM-Gal1 (1-134) with a molecular weight of 14500 Da was used forthe next step (12 ml). The fraction was concentrated with aultrafiltration unit (manufactured by Amicon; membrane YM3, 25 mm indiameter), followed by buffer exchange with 20 mM Tris-HCl buffer (pH8.0). The final solution (3 ml) was loaded at a flow rate of 0.5 ml/minat room temperature to a Shodex IEC DEAE 825 column (manufactured byShowa Denko K.K., Japan; φ8 mm×7.5 cm) equilibrated with 0% B using 20mM Tris-HCl buffer (pH 8.0) as a solvent A and the same buffercontaining 500 mM NaCl as a solvent B. After loading, the column waseluted with a 60 minutes linear gradient from 0% B to 60% B. The eluatewas fractionated into 1 ml each, and analyzed by electrophoresis. As aresult, a band that was suspected to be GIPM-Gal1 (1-134) with amolecular weight of 14500 Da was detected in a fraction withapproximately 80 mM NaCl. This fraction was used for the next step. Thefraction (1 ml) was loaded at a flow rate of 0.5 ml/min at roomtemperature to a TSKgel Phenyl-5PW RP column (manufactured by TOSOHCORPORATION, Japan; φ 4.6 mm×7.5 cm) equilibrated with 20% B, using 0.1%TFA as a solvent A and 80% acetonitrile containing 0.085% TFA as asolvent B. After loading, the column was eluted with a 40 minutes lineargradient from 20% B to 80% B. The eluate was fractionated into 1 mleach, then analyzed by electrophoresis. Only a band that was suspectedto be GIPM-Gal1 (1-134) with a molecular weight of 14500 Da was detectedin a fraction with approximately 30% acetonitirle. Thus purification wascompleted. To confirm this band is of GIPM-Gal1 (1-134), N-terminalamino acid sequence analysis was performed using a protein sequencer(manufactured by Perkin-Elmer Corporation; type 492). The detectedN-terminal amino acid sequence was as shown below (X denotesunidentified amino acids):

-   GlyIleProMetAlaXGlyLeuValAlaSerAsnLeuAsnLeu (SEQ ID NO: 62).    This sequence was identical to the N-terminal amino acid sequence of    GIPM-Gal1 (1-134) as designed. It was confirmed that the protein    purified by the TSKgel Phenyl-5PW RP column was GIPM-Gal1 (1-134).

Similarly, the clone obtained in Example 21 was expressed in E. coli,and GST-Gal1 (2/Ser) was obtained. Then the product was cleaved withfactor Xa to obtain GIMP-Gal1 (referred to as GIPM-Gal1 (2/Ser)), inwhich Cys at the 2-position was replaced by Ser.

Furthermore, the clone obtained in Example 22 was expressed in E. coli,and GST-Gal1 (all/Ser) was obtained. Then the product was cleaved withfactor Xa to obtain GIMP-Gal1 (referred to as GIPM-Gal1 (all/ser)), inwhich all of six cysteine residues was replaced by serine residues.

All references including patents and patent applications cited hereinare incorporated herein by reference in their entirety.

1. An isolated protein which possesses nerve regeneration-promotingeffect, having the amino acid sequence shown in SEQ ID NO:1 or an aminoacid sequence that has a 90% or more homology at the amino acid levelwith the amino acid sequence shown in SEQ ID NO:1, and carries adisulfide bond(s) at least between Cys at the 16-position (Cys 16) andCys at the 88-position (Cys 88) among cysteine residues at the2-position (Cys 2), 16-position (Cys 16), 42-position (Cys 42),60-position (Cys-60), 88-position (Cys 88) and 130-position (Cys 130).2. A protein of claim 1 which carries disulfide bonds between cysteineresidues of any one of combinations (1) Cys 16-Cys 88, Cys 2-Cys 130 andCys 42-Cys 60, or (2) Cys 16-Cys 88, Cys 2-Cys 60 and Cys 42-Cys 130, or(3) Cys 16-Cys 88, Cys 2-Cys 42 and Cys 60-Cys
 130. 3. A protein ofclaim 2, which comprises mixture of at least two groups out of said (1),(2), and (3).
 4. A protein of claim 3, which contains 50% or more ofsaid (1).
 5. A protein of claim 1, wherein the N-terminal end isacylated.
 6. A protein of claim 1, wherein Met⁻² Lys⁻¹ or Met⁻¹ is addedto the N-terminal end.
 7. A protein of claim 1, which is covalentlybound to a water-soluble polymer.
 8. A protein of claim 7, wherein thewater-soluble polymer is polyethylene glycol.
 9. A process for producinga protein of claim 1, comprising the steps of loading a substancecontaining said protein to an affinity column having an antibody orantibodies to said protein bound thereto, allowing said protein to beadsorbed, subsequently eluting said protein, and if necessary oxidizingsaid protein.
 10. A method for regenerating an injured nerve, comprisingadministering as the active ingredient an isolated galectin-1 thatcomprises the amino acid sequence as shown in SEQ ID No.
 1. 11. A methodfor regenerating injured nerve, comprising administering as the activeingredient a derivative of isolated galectin-1 having a 90% or morehomology at the amino acid level with the amino acid sequence shown inSEQ ID NO: 1 and possessing a nerve regeneration-promoting effect,including regeneration of axons or repair of nerve tissues.
 12. Themethod of claim 10, further comprising administering a pharmaceuticallyacceptable carrier.
 13. The method of claim 11, further comprisingadministering a pharmaceutically acceptable carrier.
 14. The method ofclaim 10, wherein said galectin-1 possesses lectin activity.
 15. Themethod of claim 10, wherein said galectin-1 possesses almost no lectinactivity or no lectin activity.
 16. The method of claim 10, wherein saidgalectin-1 has an acylated N-terminal end.
 17. The method of claim 10,wherein said galectin-1 is covalently bound to a water-soluble polymer.18. The method of claim 17, wherein said water soluble polymer ispolyethylene glycol.
 19. The method of claim 10, wherein the galectin-1carries a disulfide bond(s) at least between Cys at the 16-position (Cys16) and Cys at the ⁸⁸-position (Cys 88) among cysteine residues at the2-position (Cys 2), 16-position (Cys 16), 42-position (Cys 42),60-position (Cys-60), 88-position (Cys 88) and 130-position (Cys 130) inthe amino acid sequence shown in SEQ ID NO:1.
 20. The method of claim19, wherein the galectin-1 carries disulfide bonds between any one ofcombinations (1) Cys 16-Cys 88, Cys 2-Cys 130 and Cys 42-Cys 60, or (2)Cys 16-Cys 88, Cys 2-Cys 60 and Cys 42-Cys 130, or (3) Cys 16-Cys 88,Cys 2-Cys 42 and Cys 60-Cys
 130. 21. The method of claim 20, wherein thegalectin-1 comprises a mixture of at least two groups out of said (1),(2) and (3).
 22. The method of claim 21, wherein said mixture contains50% or more of said (1).
 23. The method of claim 10, further comprisingadministering at least one other neurotrophic factor, or paraneuralcells, or an extracellular matrix containing said factor.
 24. The methodof claim 23, wherein said extracellular matrix is laminin, collagen,fibronectin or thrombospondin.
 25. The method of claim 23, wherein saidparaneural cells are Schwann cells, fibroblasts, satellite cells,macrophage or glia cells.
 26. The method of claim 11, wherein saidgalectin-1 derivative possesses lectin activity.
 27. The method of claim11, wherein said galectin-1 derivative possesses almost no lectinactivity or no lectin activity.
 28. The method of claim 11, wherein saidgalectin-1 derivative has an acylated N-terminal end.
 29. The method ofclaim 11, wherein said galectin-1 derivative is covalently bound to awater-soluble polymer.
 30. The method of claim 11, further comprisingadministering at least one other neurotrophic factor, or paraneuralcells, or an extracellular matrix containing said factor.
 31. The methodof claim 11, Herein the galectin-1 derivative carries a disulfidebond(s) at least between Cys at the 16-position (Cys 16) and Cys at the88-position (Cys 88) among cysteine residues at the 2-position (Cys 2),16-position (Cys 16), 42-position (Cys 42), 60-position (Cys-60),88-position (Cys 88) and 130-position (Cys 130) in the amino acidsequence shown in SEQ ID NO:1.
 32. The method of claim 31, wherein thegalectin-1 derivative carries disulfide bonds between any one ofcombinations (1) Cys 16-Cys 88, Cys 2-Cys 130 and Cys 42-Cys 60, or (2)Cys 16-Cys 88, Cys 2-Cys 60 and Cys 42-Cys 130, or (3) Cys 16-Cys 88,Cys 2-Cys 42 and Cys 60-Cys
 130. 33. The method of claim 32, wherein thegalectin-1 derivative comprises a mixture of at least two groups out ofsaid (1), (2) and (3).
 34. The method of claim 30, wherein saidextracellular matrix is laminin, collagen, fibronectin orthrombospondin.
 35. The method of claim 30, wherein said paraneuralcells are Schwann cells, fibroblasts, satellite cells, macrophage orglia cells.
 36. The method of claim 28, wherein said N-terminal end ofsaid galectin-1 derivative is acetylated.
 37. The method of claim 16,wherein said N-terminal end of said galectin-1 is acetylated.
 38. Themethod of claim 29, wherein said water-soluble polymer is polyethyleneglycol.
 39. The method of claim 33, wherein said mixture contains 50% ormore of said (1).
 40. The method of claim 10, wherein said galectin-1 ispacked in a tube made of biocompatible material.
 41. The method of claim11, wherein said galectin-1 derivative is packed in biocompatiblematerial.